types of enzyme hypothesis

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Enzymes: principles and biotechnological applications

Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed.

The nature and classification of enzymes

Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms. They can also be extracted from cells and then used to catalyse a wide range of commercially important processes. For example, they have important roles in the production of sweetening agents and the modification of antibiotics, they are used in washing powders and various cleaning products, and they play a key role in analytical devices and assays that have clinical, forensic and environmental applications. The word ‘enzyme’ was first used by the German physiologist Wilhelm Kühne in 1878, when he was describing the ability of yeast to produce alcohol from sugars, and it is derived from the Greek words en (meaning ‘within’) and zume (meaning ‘yeast’).

In the late nineteenth century and early twentieth century, significant advances were made in the extraction, characterization and commercial exploitation of many enzymes, but it was not until the 1920s that enzymes were crystallized, revealing that catalytic activity is associated with protein molecules. For the next 60 years or so it was believed that all enzymes were proteins, but in the 1980s it was found that some ribonucleic acid (RNA) molecules are also able to exert catalytic effects. These RNAs, which are called ribozymes, play an important role in gene expression. In the same decade, biochemists also developed the technology to generate antibodies that possess catalytic properties. These so-called ‘abzymes’ have significant potential both as novel industrial catalysts and in therapeutics. Notwithstanding these notable exceptions, much of classical enzymology, and the remainder of this essay, is focused on the proteins that possess catalytic activity.

As catalysts, enzymes are only required in very low concentrations, and they speed up reactions without themselves being consumed during the reaction. We usually describe enzymes as being capable of catalysing the conversion of substrate molecules into product molecules as follows:

Enzymes are potent catalysts

The enormous catalytic activity of enzymes can perhaps best be expressed by a constant, k cat , that is variously referred to as the turnover rate, turnover frequency or turnover number. This constant represents the number of substrate molecules that can be converted to product by a single enzyme molecule per unit time (usually per minute or per second). Examples of turnover rate values are listed in Table 1 . For example, a single molecule of carbonic anhydrase can catalyse the conversion of over half a million molecules of its substrates, carbon dioxide (CO 2 ) and water (H 2 O), into the product, bicarbonate (HCO 3 − ), every second—a truly remarkable achievement.

Enzymes are specific catalysts

As well as being highly potent catalysts, enzymes also possess remarkable specificity in that they generally catalyse the conversion of only one type (or at most a range of similar types) of substrate molecule into product molecules.

Some enzymes demonstrate group specificity. For example, alkaline phosphatase (an enzyme that is commonly encountered in first-year laboratory sessions on enzyme kinetics) can remove a phosphate group from a variety of substrates.

Other enzymes demonstrate much higher specificity, which is described as absolute specificity. For example, glucose oxidase shows almost total specificity for its substrate, β-D-glucose, and virtually no activity with any other monosaccharides. As we shall see later, this specificity is of paramount importance in many analytical assays and devices (biosensors) that measure a specific substrate (e.g. glucose) in a complex mixture (e.g. a blood or urine sample).

Enzyme names and classification

Enzymes typically have common names (often called ‘trivial names’) which refer to the reaction that they catalyse, with the suffix -ase (e.g. oxidase, dehydrogenase, carboxylase), although individual proteolytic enzymes generally have the suffix - in (e.g. trypsin, chymotrypsin, papain). Often the trivial name also indicates the substrate on which the enzyme acts (e.g. glucose oxidase, alcohol dehydrogenase, pyruvate decarboxylase). However, some trivial names (e.g. invertase, diastase, catalase) provide little information about the substrate, the product or the reaction involved.

Due to the growing complexity of and inconsistency in the naming of enzymes, the International Union of Biochemistry set up the Enzyme Commission to address this issue. The first Enzyme Commission Report was published in 1961, and provided a systematic approach to the naming of enzymes. The sixth edition, published in 1992, contained details of nearly 3 200 different enzymes, and supplements published annually have now extended this number to over 5 000.

Within this system, all enzymes are described by a four-part Enzyme Commission (EC) number. For example, the enzyme with the trivial name lactate dehydrogenase has the EC number 1.1.1.27, and is more correctly called l –lactate: NAD + oxidoreductase.

The first part of the EC number refers to the reaction that the enzyme catalyses ( Table 2 ). The remaining digits have different meanings according to the nature of the reaction identified by the first digit. For example, within the oxidoreductase category, the second digit denotes the hydrogen donor ( Table 3 ) and the third digit denotes the hydrogen acceptor ( Table 4 ).

Thus lactate dehydrogenase with the EC number 1.1.1.27 is an oxidoreductase (indicated by the first digit) with the alcohol group of the lactate molecule as the hydrogen donor (second digit) and NAD + as the hydrogen acceptor (third digit), and is the 27th enzyme to be categorized within this group (fourth digit).

Fortunately, it is now very easy to find this information for any individual enzyme using the Enzyme Nomenclature Database (available at http://enzyme.expasy.org ).

Enzyme structure and substrate binding

Amino acid-based enzymes are globular proteins that range in size from less than 100 to more than 2 000 amino acid residues. These amino acids can be arranged as one or more polypeptide chains that are folded and bent to form a specific three-dimensional structure, incorporating a small area known as the active site ( Figure 1 ), where the substrate actually binds. The active site may well involve only a small number (less than 10) of the constituent amino acids.

It is the shape and charge properties of the active site that enable it to bind to a single type of substrate molecule, so that the enzyme is able to demonstrate considerable specificity in its catalytic activity.

The hypothesis that enzyme specificity results from the complementary nature of the substrate and its active site was first proposed by the German chemist Emil Fischer in 1894, and became known as Fischer's ‘lock and key hypothesis’, whereby only a key of the correct size and shape (the substrate) fits into the keyhole (the active site) of the lock (the enzyme). It is astounding that this theory was proposed at a time when it was not even established that enzymes were proteins. As more was learned about enzyme structure through techniques such as X-ray crystallography, it became clear that enzymes are not rigid structures, but are in fact quite flexible in shape. In the light of this finding, in 1958 Daniel Koshland extended Fischer's ideas and presented the ‘induced-fit model’ of substrate and enzyme binding, in which the enzyme molecule changes its shape slightly to accommodate the binding of the substrate. The analogy that is commonly used is the ‘hand-in-glove model’, where the hand and glove are broadly complementary in shape, but the glove is moulded around the hand as it is inserted in order to provide a perfect match.

Since it is the active site alone that binds to the substrate, it is logical to ask what is the role of the rest of the protein molecule. The simple answer is that it acts to stabilize the active site and provide an appropriate environment for interaction of the site with the substrate molecule. Therefore the active site cannot be separated out from the rest of the protein without loss of catalytic activity, although laboratory-based directed (or forced) evolution studies have shown that it is sometimes possible to generate smaller enzymes that do retain activity.

It should be noted that although a large number of enzymes consist solely of protein, many also contain a non-protein component, known as a cofactor, that is necessary for the enzyme's catalytic activity. A cofactor may be another organic molecule, in which case it is called a coenzyme, or it may be an inorganic molecule, typically a metal ion such as iron, manganese, cobalt, copper or zinc. A coenzyme that binds tightly and permanently to the protein is generally referred to as the prosthetic group of the enzyme.

When an enzyme requires a cofactor for its activity, the inactive protein component is generally referred to as an apoenzyme, and the apoenzyme plus the cofactor (i.e. the active enzyme) is called a holoenzyme ( Figure 2 ).

The need for minerals and vitamins in the human diet is partly attributable to their roles within metabolism as cofactors and coenzymes.

Enzymes and reaction equilibrium

How do enzymes work? The broad answer to this question is that they do not alter the equilibrium (i.e. the thermodynamics) of a reaction. This is because enzymes do not fundamentally change the structure and energetics of the products and reagents, but rather they simply allow the reaction equilibrium to be attained more rapidly. Let us therefore begin by clarifying the concept of chemical equilibrium.

In many cases the equilibrium of a reaction is far ‘to the right’—that is, virtually all of the substrate (S) is converted into product (P). For this reason, reactions are often written as follows:

This is a simplification, as in all cases it is more correct to write this reaction as follows:

This indicates the presence of an equilibrium. To understand this concept it is perhaps most helpful to look at a reaction where the equilibrium point is quite central.

For example:

In this reaction, if we start with a solution of 1 mol l −1 glucose and add the enzyme, then upon completion we will have a mixture of approximately 0.5 mol l −1 glucose and 0.5 mol l −1 fructose. This is the equilibrium point of this particular reaction, and although it may only take a couple of seconds to reach this end point with the enzyme present, we would in fact come to the same point if we put glucose into solution and waited many months for the reaction to occur in the absence of the enzyme. Interestingly, we could also have started this reaction with a 1 mol l −1 fructose solution, and it would have proceeded in the opposite direction until the same equilibrium point had been reached.

The equilibrium point for this reaction is expressed by the equilibrium constant K eq as follows:

Thus for a reaction with central equilibrium, K eq = 1, for an equilibrium ‘to the right’ K eq is >1, and for an equilibrium ‘to the left’ K eq is <1.

Therefore if a reaction has a K eq value of 10 6 , the equilibrium is very far to the right and can be simplified by denoting it as a single arrow. We may often describe this type of reaction as ‘going to completion’. Conversely, if a reaction has a K eq value of 10 −6 , the equilibrium is very far to the left, and for all practical purposes it would not really be considered to proceed at all.

It should be noted that although the concentration of reactants has no effect on the equilibrium point, environmental factors such as pH and temperature can and do affect the position of the equilibrium.

It should also be noted that any biochemical reaction which occurs in vivo in a living system does not occur in isolation, but as part of a metabolic pathway, which makes it more difficult to conceptualize the relationship between reactants and reactions. In vivo reactions are not allowed to proceed to their equilibrium position. If they did, the reaction would essentially stop (i.e. the forward and reverse reactions would balance each other), and there would be no net flux through the pathway. However, in many complex biochemical pathways some of the individual reaction steps are close to equilibrium, whereas others are far from equilibrium, the latter (catalysed by regulatory enzymes) having the greatest capacity to control the overall flux of materials through the pathway.

Enzymes form complexes with their substrates

We often describe an enzyme-catalysed reaction as proceeding through three stages as follows:

The ES complex represents a position where the substrate (S) is bound to the enzyme (E) such that the reaction (whatever it might be) is made more favourable. As soon as the reaction has occurred, the product molecule (P) dissociates from the enzyme, which is then free to bind to another substrate molecule. At some point during this process the substrate is converted into an intermediate form (often called the transition state) and then into the product.

The exact mechanism whereby the enzyme acts to increase the rate of the reaction differs from one system to another. However, the general principle is that by binding of the substrate to the enzyme, the reaction involving the substrate is made more favourable by lowering the activation energy of the reaction.

In terms of energetics, reactions can be either exergonic (releasing energy) or endergonic (consuming energy). However, even in an exergonic reaction a small amount of energy, termed the activation energy, is needed to give the reaction a ‘kick start.’ A good analogy is that of a match, the head of which contains a mixture of energy-rich chemicals (phosphorus sesquisulfide and potassium chlorate). When a match burns it releases substantial amounts of light and heat energy (exergonically reacting with O 2 in the air). However, and perhaps fortunately, a match will not spontaneously ignite, but rather a small input of energy in the form of heat generated through friction (i.e. striking of the match) is needed to initiate the reaction. Of course once the match has been struck the amount of energy released is considerable, and greatly exceeds the small energy input during the striking process.

As shown in Figure 3 , enzymes are considered to lower the activation energy of a system by making it energetically easier for the transition state to form. In the presence of an enzyme catalyst, the formation of the transition state is energetically more favourable (i.e. it requires less energy for the ‘kick start’), thereby accelerating the rate at which the reaction will proceed, but not fundamentally changing the energy levels of either the reactant or the product.

Properties and mechanisms of enzyme action

Enzyme kinetics.

Enzyme kinetics is the study of factors that determine the speed of enzyme-catalysed reactions. It utilizes some mathematical equations that can be confusing to students when they first encounter them. However, the theory of kinetics is both logical and simple, and it is essential to develop an understanding of this subject in order to be able to appreciate the role of enzymes both in metabolism and in biotechnology.

Assays (measurements) of enzyme activity can be performed in either a discontinuous or continuous fashion. Discontinuous methods involve mixing the substrate and enzyme together and measuring the product formed after a set period of time, so these methods are generally easy and quick to perform. In general we would use such discontinuous assays when we know little about the system (and are making preliminary investigations), or alternatively when we know a great deal about the system and are certain that the time interval we are choosing is appropriate.

In continuous enzyme assays we would generally study the rate of an enzyme-catalysed reaction by mixing the enzyme with the substrate and continuously measuring the appearance of product over time. Of course we could equally well measure the rate of the reaction by measuring the disappearance of substrate over time. Apart from the actual direction (one increasing and one decreasing), the two values would be identical. In enzyme kinetics experiments, for convenience we very often use an artificial substrate called a chromogen that yields a brightly coloured product, making the reaction easy to follow using a colorimeter or a spectrophotometer. However, we could in fact use any available analytical equipment that has the capacity to measure the concentration of either the product or the substrate.

In almost all cases we would also add a buffer solution to the mixture. As we shall see, enzyme activity is strongly influenced by pH, so it is important to set the pH at a specific value and keep it constant throughout the experiment.

Our first enzyme kinetics experiment may therefore involve mixing a substrate solution (chromogen) with a buffer solution and adding the enzyme. This mixture would then be placed in a spectrophotometer and the appearance of the coloured product would be measured. This would enable us to follow a rapid reaction which, after a few seconds or minutes, might start to slow down, as shown in Figure 4 .

A common reason for this slowing down of the speed (rate) of the reaction is that the substrate within the mixture is being used up and thus becoming limiting. Alternatively, it may be that the enzyme is unstable and is denaturing over the course of the experiment, or it could be that the pH of the mixture is changing, as many reactions either consume or release protons. For these reasons, when we are asked to specify the rate of a reaction we do so early on, as soon as the enzyme has been added, and when none of the above-mentioned limitations apply. We refer to this initial rapid rate as the initial velocity ( v 0 ). Measurement of the reaction rate at this early stage is also quite straightforward, as the rate is effectively linear, so we can simply draw a straight line and measure the gradient (by dividing the concentration change by the time interval) in order to evaluate the reaction rate over this period.

We may now perform a range of similar enzyme assays to evaluate how the initial velocity changes when the substrate or enzyme concentration is altered, or when the pH is changed. These studies will help us to characterize the properties of the enzyme under study.

The relationship between enzyme concentration and the rate of the reaction is usually a simple one. If we repeat the experiment just described, but add 10% more enzyme, the reaction will be 10% faster, and if we double the enzyme concentration the reaction will proceed twice as fast. Thus there is a simple linear relationship between the reaction rate and the amount of enzyme available to catalyse the reaction ( Figure 5 ).

This relationship applies both to enzymes in vivo and to those used in biotechnological applications, where regulation of the amount of enzyme present may control reaction rates.

When we perform a series of enzyme assays using the same enzyme concentration, but with a range of different substrate concentrations, a slightly more complex relationship emerges, as shown in Figure 6 . Initially, when the substrate concentration is increased, the rate of reaction increases considerably. However, as the substrate concentration is increased further the effects on the reaction rate start to decline, until a stage is reached where increasing the substrate concentration has little further effect on the reaction rate. At this point the enzyme is considered to be coming close to saturation with substrate, and demonstrating its maximal velocity ( V max ). Note that this maximal velocity is in fact a theoretical limit that will not be truly achieved in any experiment, although we might come very close to it.

The relationship described here is a fairly common one, which a mathematician would immediately identify as a rectangular hyperbola. The equation that describes such a relationship is as follows:

The two constants a and b thus allow us to describe this hyperbolic relationship, just as with a linear relationship ( y = mx + c ), which can be expressed by the two constants m (the slope) and c (the intercept).

We have in fact already defined the constant a — it is V max . The constant b is a little more complex, as it is the value on the x -axis that gives half of the maximal value of y . In enzymology we refer to this as the Michaelis constant ( K m ), which is defined as the substrate concentration that gives half-maximal velocity.

Our final equation, usually called the Michaelis–Menten equation, therefore becomes:

In 1913, Leonor Michaelis and Maud Menten first showed that it was in fact possible to derive this equation mathematically from first principles, with some simple assumptions about the way in which an enzyme reacts with a substrate to form a product. Central to their derivation is the concept that the reaction takes place via the formation of an ES complex which, once formed, can either dissociate (productively) to release product, or else dissociate in the reverse direction without any formation of product. Thus the reaction can be represented as follows, with k 1 , k −1 and k 2 being the rate constants of the three individual reaction steps:

The Michaelis–Menten derivation requires two important assumptions. The first assumption is that we are considering the initial velocity of the reaction ( v 0 ), when the product concentration will be negligibly small (i.e. [S] ≫ [P]), such that we can ignore the possibility of any product reverting to substrate. The second assumption is that the concentration of substrate greatly exceeds the concentration of enzyme (i.e. [S]≫[E]).

The derivation begins with an equation for the expression of the initial rate, the rate of formation of product, as the rate at which the ES complex dissociates to form product. This is based upon the rate constant k 2 and the concentration of the ES complex, as follows:

Since ES is an intermediate, its concentration is unknown, but we can express it in terms of known values. In a steady-state approximation we can assume that although the concentration of substrate and product changes, the concentration of the ES complex itself remains constant. The rate of formation of the ES complex and the rate of its breakdown must therefore balance, where:

Hence, at steady state:

This equation can be rearranged to yield [ES] as follows:

The Michaelis constant K m can be defined as follows:

Equation 2 may thus be simplified to:

Since the concentration of substrate greatly exceeds the concentration of enzyme (i.e. [S] ≫ [E]), the concentration of uncombined substrate [S] is almost equal to the total concentration of substrate. The concentration of uncombined enzyme [E] is equal to the total enzyme concentration [E] T minus that combined with substrate [ES]. Introducing these terms to Equation 3 and solving for ES gives us the following:

We can then introduce this term into Equation 1 to give:

The term k 2 [E] T in fact represents V max , the maximal velocity. Thus Michaelis and Menten were able to derive their final equation as:

A more detailed derivation of the Michaelis–Menten equation can be found in many biochemistry textbooks (see section 4 of Recommended Reading section). There are also some very helpful web-based tutorials available on the subject.

Michaelis constants have been determined for many commonly used enzymes, and are typically in the lower millimolar range ( Table 5 ).

It should be noted that enzymes which catalyse the same reaction, but which are derived from different organisms, can have widely differing K m values. Furthermore, an enzyme with multiple substrates can have quite different K m values for each substrate.

A low K m value indicates that the enzyme requires only a small amount of substrate in order to become saturated. Therefore the maximum velocity is reached at relatively low substrate concentrations. A high K m value indicates the need for high substrate concentrations in order to achieve maximum reaction velocity. Thus we generally refer to K m as a measure of the affinity of the enzyme for its substrate—in fact it is an inverse measure, where a high K m indicates a low affinity, and vice versa.

The K m value tells us several important things about a particular enzyme.

• An enzyme with a low K m value relative to the physiological concentration of substrate will probably always be saturated with substrate, and will therefore act at a constant rate, regardless of variations in the concentration of substrate within the physiological range.
• An enzyme with a high K m value relative to the physiological concentration of substrate will not be saturated with substrate, and its activity will therefore vary according to the concentration of substrate, so the rate of formation of product will depend on the availability of substrate.
• If an enzyme acts on several substrates, the substrate with the lowest K m value is frequently assumed to be that enzyme's ‘natural’ substrate, although this may not be true in all cases.
• If two enzymes (with similar V max ) in different metabolic pathways compete for the same substrate, then if we know the K m values for the two enzymes we can predict the relative activity of the two pathways. Essentially the pathway that has the enzyme with the lower K m value is likely to be the ‘preferred pathway’, and more substrate will flow through that pathway under most conditions. For example, phosphofructokinase (PFK) is the enzyme that catalyses the first committed step in the glycolytic pathway, which generates energy in the form of ATP for the cell, whereas glucose-1-phosphate uridylyltransferase (GUT) is an enzyme early in the pathway leading to the synthesis of glycogen (an energy storage molecule). Both enzymes use hexose monophosphates as substrates, but the K m of PFK for its substrate is lower than that of GUT for its substrate. Thus at lower cellular hexose phosphate concentrations, PFK will be active and GUT will be largely inactive. At higher hexose phosphate concentrations both pathways will be active. This means that the cells only store glycogen in times of plenty, and always give preference to the pathway of ATP production, which is the more essential function.

Very often it is not possible to estimate K m values from a direct plot of velocity against substrate concentration (as shown in Figure 6 ) because we have not used high enough substrate concentrations to come even close to estimating maximal velocity, and therefore we cannot evaluate half-maximal velocity and thus K m . Fortunately, we can plot our experimental data in a slightly different way in order to obtain these values. The most commonly used alternative is the Lineweaver–Burk plot (often called the double-reciprocal plot). This plot linearizes the hyperbolic curved relationship, and the line produced is easy to extrapolate, allowing evaluation of V max and K m . For example, if we obtained only the first seven data points in Figure 6 , we would have difficulty estimating V max from a direct plot as shown in Figure 7 a.

However, as shown in Figure 7 b, if these seven points are plotted on a graph of 1/velocity against 1/substrate concentration (i.e. a double-reciprocal plot), the data are linearized, and the line can be easily extrapolated to the left to provide intercepts on both the y -axis and the x -axis, from which V max and K m , respectively, can be evaluated.

One significant practical drawback of using the Lineweaver–Burk plot is the excessive influence that it gives to measurements made at the lowest substrate concentrations. These concentrations might well be the most prone to error (due to difficulties in making multiple dilutions), and result in reaction rates that, because they are slow, might also be most prone to measurement error. Often, as shown in Figure 8 , such points when transformed on the Lineweaver–Burk plot have a significant impact on the line of best fit estimated from the data, and therefore on the extrapolated values of both V max and K m . The two sets of points shown in Figure 8 are identical except for the single point at the top right, which reflects (because of the plot's double-reciprocal nature) a single point derived from a very low substrate concentration and a low reaction rate. However, this single point can have an enormous impact on the line of best fit and the accompanying estimates of kinetic constants.

In fact there are other kinetic plots that can be used, including the Eadie–Hofstee plot, the Hanes plot and the Eisenthal–Cornish-Bowden plot, which are less prone to such problems. However, the Lineweaver–Burk plot is still the most commonly described kinetic plot in the majority of enzymology textbooks, and thus retains its influence in undergraduate education.

Enzymes are affected by pH and temperature

Various environmental factors are able to affect the rate of enzyme-catalysed reactions through reversible or irreversible changes in the protein structure. The effects of pH and temperature are generally well understood.

Most enzymes have a characteristic optimum pH at which the velocity of the catalysed reaction is maximal, and above and below which the velocity declines ( Figure 9 ).

The pH profile is dependent on a number of factors. As the pH changes, the ionization of groups both at the enzyme's active site and on the substrate can alter, influencing the rate of binding of the substrate to the active site. These effects are often reversible. For example, if we take an enzyme with an optimal pH (pH opt ) of 7.0 and place it in an environment at pH 6.0 or 8.0, the charge properties of the enzyme and the substrate may be suboptimal, such that binding and hence the reaction rate are lowered. If we then readjust the pH to 7.0, the optimal charge properties and hence the maximal activity of the enzyme are often restored. However, if we place the enzyme in a more extreme acidic or alkaline environment (e.g. at pH 1 or 14), although these conditions may not actually lead to changes in the very stable covalent structure of the protein (i.e. its configuration), they may well produce changes in the conformation (shape) of the protein such that, when it is returned to pH 7.0, the original conformation and hence the enzyme's full catalytic activity are not restored.

It should be noted that the optimum pH of an enzyme may not be identical to that of its normal intracellular surroundings. This indicates that the local pH can exert a controlling influence on enzyme activity.

The effects of temperature on enzyme activity are quite complex, and can be regarded as two forces acting simultaneously but in opposite directions. As the temperature is raised, the rate of molecular movement and hence the rate of reaction increases, but at the same time there is a progressive inactivation caused by denaturation of the enzyme protein. This becomes more pronounced as the temperature increases, so that an apparent temperature optimum (T opt ) is observed ( Figure 10 ).

Thermal denaturation is time dependent, and for an enzyme the term ‘optimum temperature’ has little real meaning unless the duration of exposure to that temperature is recorded. The thermal stability of an enzyme can be determined by first exposing the protein to a range of temperatures for a fixed period of time, and subsequently measuring its activity at one favourable temperature (e.g. 25°C).

The temperature at which denaturation becomes important varies from one enzyme to another. Normally it is negligible below 30°C, and starts to become appreciable above 40°C. Typically, enzymes derived from microbial sources show much higher thermal stability than do those from mammalian sources, and enzymes derived from extremely thermophilic microorganisms, such as thermolysin (a protease from Bacillus thermoproteolyticus ) and Taq polymerase (a DNA polymerase from Thermus aquaticus ), might be completely thermostable at 70°C and still retain substantial levels of activity even at 100°C.

Enzymes are sensitive to inhibitors

Substances that reduce the activity of an enzyme-catalysed reaction are known as inhibitors. They act by either directly or indirectly influencing the catalytic properties of the active site. Inhibitors can be foreign to the cell or natural components of it. Those in the latter category can represent an important element of the regulation of cell metabolism. Many toxins and also many pharmacologically active agents (both illegal drugs and prescription and over-the-counter medicines) act by inhibiting specific enzyme-catalysed processes.

Reversible inhibition

Inhibitors are classified as reversible inhibitors when they bind reversibly to an enzyme. A molecule that is structurally similar to the normal substrate may be able to bind reversibly to the enzyme's active site and therefore act as a competitive inhibitor. For example, malonate is a competitive inhibitor of the enzyme succinate dehydrogenase, as it is capable of binding to the enzyme's active site due to its close structural similarity to the enzyme's natural substrate, succinate (see below). When malonate occupies the active site of succinate dehydrogenase it prevents the natural substrate, succinate, from binding, thereby slowing down the rate of oxidation of succinate to fumarate (i.e. inhibiting the reaction).

One of the characteristics of competitive inhibitors is that they can be displaced from the active site if high concentrations of substrate are used, thereby restoring enzyme activity. Thus competitive inhibitors increase the K m of a reaction because they increase the concentration of substrate required to saturate the enzyme. However, they do not change V max itself.

In the case of certain enzymes, high concentrations of either the substrate or the product can be inhibitory. For example, invertase activity is considerably reduced in the presence of high concentrations of sucrose (its substrate), whereas the β-galactosidase of Aspergillus niger is strongly inhibited by galactose (its product). Products of an enzyme reaction are some of the most commonly encountered competitive inhibitors.

Other types of reversible inhibitor also exist. Non-competitive inhibitors react with the enzyme at a site distinct from the active site. Therefore the binding of the inhibitor does not physically block the substrate–binding site, but it does prevent subsequent reaction. Most non-competitive inhibitors are chemically unrelated to the substrate, and their inhibition cannot be overcome by increasing the substrate concentration. Such inhibitors in effect reduce the concentration of the active enzyme in solution, thereby reducing the V max of the reaction. However, they do not change the value of K m .

Uncompetitive inhibition is rather rare, occurring when the inhibitor is only able to bind to the enzyme once a substrate molecule has itself bound. As such, inhibition is most significant at high substrate concentrations, and results in a reduction in the V max of the reaction. Uncompetitive inhibition also causes a reduction in K m , which seems somewhat counterintuitive as this means that the affinity of the enzyme for its substrate is actually increased when the inhibitor is present. This effect occurs because the binding of the inhibitor to the ES complex effectively removes ES complex and thereby affects the overall equilibrium of the reaction favouring ES complex formation. It is noteworthy however that since both V max and K m are reduced the observed reaction rates with inhibitor present are always lower than those in the absence of the uncompetitive inhibitor.

Irreversible inhibitors and poisons

If an inhibitor binds permanently to an enzyme it is known as an irreversible inhibitor. Many irreversible inhibitors are therefore potent toxins.

Organophosphorus compounds such as diisopropyl fluorophosphate (DFP) inhibit acetylcholinesterase activity by reacting covalently with an important serine residue found within the active site of the enzyme. The physiological effect of this inactivation is interference with neurotransmitter inactivation at the synapses of nerves, resulting in the constant propagation of nerve impulses, which can lead to death. DFP was originally evaluated by the British as a chemical warfare agent during World War Two, and modified versions of this compound are now widely used as organophosphate pesticides (e.g. parathione, malathione).

Allosteric regulators and the control of enzyme activity

Having spent time learning about enzyme kinetics and the Michaelis–Menten relationship, it is often quite disconcerting to find that some of the most important enzymes do not in fact display such properties. Allosteric enzymes are key regulatory enzymes that control the activities of metabolic pathways by responding to inhibitors and activators. These enzymes in fact show a sigmoidal (S-shaped) relationship between reaction rate and substrate concentration ( Figure 11 ), rather than the usual hyperbolic relationship. Thus for allosteric enzymes there is an area where activity is lower than that of an equivalent ‘normal’ enzyme, and also an area where activity is higher than that of an equivalent ‘normal’ enzyme, with a rapid transition between these two phases. This is rather like a switch that can quickly be changed from ‘off’ (low activity) to ‘on’ (full activity).

Most allosteric enzymes are polymeric—that is, they are composed of at least two (and often many more) individual polypeptide chains. They also have multiple active sites where the substrate can bind. Much of our understanding of the function of allosteric enzymes comes from studies of haemoglobin which, although it is not an enzyme, binds oxygen in a similarly co-operative way and thus also demonstrates this sigmoidal relationship. Allosteric enzymes have an initially low affinity for the substrate, but when a single substrate molecule binds, this may break some bonds within the enzyme and thereby change the shape of the protein such that the remaining active sites are able to bind with a higher affinity. Therefore allosteric enzymes are often described as moving from a tensed state or T-state (low affinity) in which no substrate is bound, to a relaxed state or R-state (high affinity) as substrate binds. Other molecules can also bind to allosteric enzymes, at additional regulatory sites (i.e. not at the active site). Molecules that stabilize the protein in its T-state therefore act as allosteric inhibitors, whereas molecules that move the protein to its R-state will act as allosteric activators or promoters.

A good example of an allosteric enzyme is aspartate transcarbamoylase (ATCase), a key regulatory enzyme that catalyses the first committed step in the sequence of reactions that produce the pyrimidine nucleotides which are essential components of DNA and RNA. The reaction is as follows:

The end product in the pathway, the pyrimidine nucleotide cytidine triphosphate (CTP), is an active allosteric inhibitor of the enzyme ATCase. Therefore when there is a high concentration of CTP in the cell, this feeds back and inhibits the ATCase enzyme, reducing its activity and thus lowering the rate of production of further pyrimidine nucleotides. As the concentration of CTP in the cell decreases then so does the inhibition of ATCase, and the resulting increase in enzyme activity leads to the production of more pyrimidine nucleotides. This negative feedback inhibition is an important element of biochemical homeostasis within the cell. However, in order to synthesize DNA and RNA, the cell requires not only pyrimidine nucleotides but also purine nucleotides, and these are needed in roughly equal proportions. Purine synthesis occurs through a different pathway, but interestingly the final product, the purine nucleotide adenosine triphosphate (ATP), is a potent activator of the enzyme ATCase. This is logical, since when the cell contains high concentrations of purine nucleotides it will require equally high concentrations of pyrimidine nucleotides in order for these two types of nucleotide to combine to form the polymers DNA and RNA. Thus ATCase is able to regulate the production of pyrimidine nucleotides within the cell according to cellular demand, and also to ensure that pyrimidine nucleotide synthesis is synchronized with purine nucleotide synthesis—an elegant biochemical mechanism for the regulation of an extremely important metabolic process.

There are some rare, although important, cases of monomeric enzymes that have only one substrate-binding site but are capable of demonstrating the sigmoidal reaction kinetics characteristic of allosteric enzymes. Particularly noteworthy in this context is the monomeric enzyme glucokinase (also called hexokinase IV), which catalyses the phosphorylation of glucose to glucose-6-phosphate (which may then either be metabolized by the glycolytic pathway or be used in glycogen synthesis). It has been postulated that this kinetic behaviour is a result of individual glucokinase molecules existing in one of two forms—a low-affinity form and a high-affinity form. The low-affinity form of the enzyme reacts with its substrate (glucose), is then turned into the high-affinity form, and remains in that state for a short time before slowly returning to its original low-affinity form (demonstrating a so-called slow transition). Therefore at high substrate concentrations the enzyme is likely to react with a second substrate molecule soon after the first one (i.e. while still in its high-affinity form), whereas at lower substrate concentrations the enzyme may transition back to its low-affinity form before it reacts with subsequent substrate molecules. This results in its characteristic sigmoidal reaction kinetics.

Origin, purification and uses of enzymes

Enzymes are ubiquitous.

Enzymes are essential components of animals, plants and microorganisms, due to the fact that they catalyse and co-ordinate the complex reactions of cellular metabolism.

Up until the 1970s, most of the commercial application of enzymes involved animal and plant sources. At that time, bulk enzymes were generally only used within the food-processing industry, and enzymes from animals and plants were preferred, as they were considered to be free from the problems of toxicity and contamination that were associated with enzymes of microbial origin. However, as demand grew and as fermentation technology developed, the competitive cost of microbial enzymes was recognized and they became more widely used.

Compared with enzymes from plant and animal sources, microbial enzymes have economic, technical and ethical advantages, which will now be outlined.

Economic advantages

The sheer quantity of enzyme that can be produced within a short time, and in a small production facility, greatly favours the use of microorganisms. For example, during the production of rennin (a milk-coagulating enzyme used in cheese manufacture) the traditional approach is to use the enzyme extracted from the stomach of a calf (a young cow still feeding on its mother's milk). The average quantity of rennet extracted from a calf's stomach is 10 kg, and it takes several months of intensive farming to produce a calf. In comparison, a 1 000-litre fermenter of recombinant Bacillus subtilis can produce 20 kg of enzyme within 12 h. Thus the microbial product is clearly preferable economically, and is free from the ethical issues that surround the use of animals. Indeed, most of the cheese now sold in supermarkets is made from milk coagulated with microbial enzymes (so is suitable for vegetarians).

A further advantage of using microbial enzymes is their ease of extraction. Many of the microbial enzymes used in biotechnological processes are secreted extracellularly, which greatly simplifies their extraction and purification. Microbial intracellular enzymes are also often easier to obtain than the equivalent animal or plant enzymes, as they generally require fewer extraction and purification steps.

Animal and plant sources usually need to be transported to the extraction facility, whereas when microorganisms are used the same facility can generally be employed for production and extraction. In addition, commercially important animal and plant enzymes are often located within only one organ or tissue, so the remaining material is essentially a waste product, disposal of which is required.

Finally, enzymes from plant and animal sources show wide variation in yield, and may only be available at certain times of year, whereas none of these problems are associated with microbial enzymes.

Technical advantages

Microbial enzymes often have properties that make them more suitable for commercial exploitation. In comparison with enzymes from animal and plant sources, the stability of microbial enzymes is usually high. For example, the high temperature stability of enzymes from thermophilic microorganisms is often useful when the process must operate at high temperatures (e.g. during starch processing).

Microorganisms are also very amenable to genetic modification to produce novel or altered enzymes, using relatively simple methods such as plasmid insertion. The genetic manipulation of animals and plants is technically much more difficult, is more expensive and is still the subject of significant ethical concern, especially in the U.K.

Enzymes may be intracellular or extracellular

Although many enzymes are retained within the cell, and may be located in specific subcellular compartments, others are released into the surrounding environment. The majority of enzymes in industrial use are extracellular proteins from either fungal sources (e.g. Aspergillus species) or bacterial sources (e.g. Bacillus species). Examples of these include α-amylase, cellulase, dextranase, proteases and amyloglucosidase. Many other enzymes for non-industrial use are intracellular and are produced in much smaller amounts by the cell. Examples of these include asparaginase, catalase, cholesterol oxidase, glucose oxidase and glucose-6-phosphate dehydrogenase.

Enzyme purification

Within the cell, enzymes are generally found along with other proteins, nucleic acids, polysaccharides and lipids. The activity of the enzyme in relation to the total protein present (i.e. the specific activity) can be determined and used as a measure of enzyme purity. A variety of methods can be used to remove contaminating material in order to purify the enzyme and increase its specific activity. Enzymes that are used as diagnostic reagents and in clinical therapeutics are normally prepared to a high degree of purity, because great emphasis is placed on the specificity of the reaction that is being catalysed. Clearly the higher the level of purification, the greater the cost of enzyme production. In the case of many bulk industrial enzymes the degree of purification is less important, and such enzymes may often be sold as very crude preparations of culture broth containing the growth medium, organisms (whole or fragmented) and enzymes of interest. However, even when the cheapest bulk enzymes are utilized (e.g. proteases for use in washing powders), the enzyme cost can contribute around 5–10% of the final product value.

Pretreatment

At the end of a fermentation in which a microorganism rich in the required enzyme has been cultured, the broth may be cooled rapidly to 5°C to prevent further microbial growth and stabilize the enzyme product. The pH may also be adjusted to optimize enzyme stability. If the enzyme-producing organism is a fungus, this may be removed by centrifugation at low speed. If the enzyme source is bacterial, the bacteria are often flocculated with aluminum sulfate or calcium chloride, which negate the charge on the bacterial membranes, causing them to clump and thus come out of suspension.

Extracellular enzymes are found in the liquid component of the pretreatment process. However, intracellular enzymes require more extensive treatment. The biomass may be concentrated by centrifugation and washed to remove medium components. The cellular component must then be ruptured to release the enzyme content. This can be done using one or more of the following processes:

• • ball milling (using glass beads)
• • enzymic removal of the cell wall
• • freeze–thaw cycles
• • liquid shearing through a small orifice at high pressure (e.g. within a French press)
• • osmotic shock
• • sonication.

Separation of enzymes from the resulting solution may then involve a variety of separation processes, which are often employed in a sequential fashion.

The first step in an enzyme purification procedure commonly involves separation of the proteins from the non-protein components by a process of salting out. Proteins remain in aqueous solution because of interactions between the hydrophilic (water-loving) amino acids and the surrounding water molecules (the solvent). If the ionic strength of the solvent is increased by adding an agent such as ammonium sulfate, some of the water molecules will interact with the salt ions, thereby decreasing the number of water molecules available to interact with the protein. Under such conditions, when protein molecules cannot interact with the solvent, they interact with each other, coagulating and coming out of solution in the form of a precipitate. This precipitate (containing the enzyme of interest and other proteins) can then be filtered or centrifuged, and separated from the supernatant.

Since different proteins vary in the extent to which they interact with water, it is possible to perform this process using a series of additions of ammonium sulfate, increasing the ionic strength in a stepwise fashion and removing the precipitate at each stage. Thus such fractional precipitation is not only capable of separating protein from non-protein components, but can also enable separation of the enzyme of interest from some of the other protein components.

Subsequently a wide variety of techniques may be used for further purification, and steps involving chromatography are standard practice.

Ion-exchange chromatography is often effective during the early stages of the purification process. The protein solution is added to a column containing an insoluble polymer (e.g. cellulose) that has been modified so that its ionic characteristics will determine the type of mobile ion (i.e. cation or anion) it attracts. Proteins whose net charge is opposite to that of the ion-exchange material will bind to it, whereas all other proteins will pass through the column. A subsequent change in pH or the introduction of a salt solution will alter the electrostatic forces, allowing the retained protein to be released into solution again.

Gel filtration can be utilized in the later stages of a purification protocol to separate molecules on the basis of molecular size. Columns containing a bed of cross-linked gel particles such as Sephadex are used. These gel particles exclude large protein molecules while allowing the entry of smaller molecules. Separation occurs because the larger protein molecules follow a path down the column between the Sephadex particles (occupying a smaller fraction of the column volume). Larger molecules therefore have a shorter elution time and are recovered first from the gel filtration column.

Affinity chromatography procedures can often enable purification protocols to be substantially simplified. Typically, with respect to enzyme purification, a column would be packed with a particulate stationary phase to which a ligand molecule such as a substrate analogue, inhibitor or cofactor of the enzyme of interest would be firmly bound. As the sample mixture is passed through the column, the enzyme interacts with, and binds, to the immobilised ligand, being retained within the column as all of the other components of the mixture pass through the column unrewarded. Subsequently a solution of the ligand is introduced to the column to release (elute) and thereby recover the bound enzyme from the column in a highly purified form.

Nowadays numerous alternative affinity chromatography procedures exist that are able to separate enzymes by binding to areas of the molecule away form their active site. Advances in molecular biology enable us to purify recombinant proteins, including enzymes, through affinity tagging. In a typical approach the gene for the enzyme of interest would be modified to code for a further short amino acid sequence at either the N- or C- terminal. For example, a range of polyhistidine tagging procedures are available to yield protein products with six or more consecutive histidine residues at their N- or C- terminal end. When a mixture containing the tagged protein of interest is subsequently passed through a column containing a nickel-nitrilotriacetic acid (Ni-NTA) agarose resin, the histidine residues on the recombinant protein bind to the nickel ions attached to the support resin, retaining the protein, whilst other protein and non-protein components pass through the column. Elution of the bound protein can then be accomplished by adding imidazole to the column, or by reducing the pH to 5-6 to displace the His-tagged protein from the nickel ions.

Such techniques are therefore capable of rapidly and highly effectively isolating an enzyme from a complex mixture in only one step, and typically provide protein purities of up to 95%. If more highly purified enzyme products are required, other supplemental options are also available, including various forms of preparative electrophoresis e.g. disc-gel electrophoresis and isoelectric focusing.

Finishing of enzymes

Enzymes are antigenic, and since problems occurred in the late 1960s when manufacturing workers exhibited severe allergic responses after breathing enzyme dusts, procedures have now been implemented to reduce dust formation. These involve supplying enzymes as liquids wherever possible, or increasing the particle size of dry powders from 10 μm to 200–500 μm by either prilling (mixing the enzyme with polyethylene glycol and preparing small spheres by atomization) or marumerizing (mixing the enzyme with a binder and water, extruding long filaments, converting them into spheres in a marumerizer, drying them and covering them with a waxy coat).

Industrial enzymology

Although many industrial processes, such as cheese manufacturing, have traditionally used impure enzyme sources, often from animals or plants, the development of much of modern industrial enzymology has gone hand in hand with the commercial exploitation of microbial enzymes. These were introduced to the West in around 1890 when the Japanese scientist Jokichi Takamine settled in the U.S.A. and set up an enzyme factory based on Japanese technology. The principal product was Takadiastase, a mixture of amylolytic and proteolytic enzymes prepared by cultivating the fungus Aspergillus oryzae on rice or wheat bran. Takadiastase was marketed successfully in the U.S.A. as a digestive aid for the treatment of dyspepsia, which was then believed to result from the incomplete digestion of starch.

Bacterial enzymes were developed in France by August Boidin and Jean Effront, who in 1913 found that Bacillus subtilis produced a heat-stable α-amylase when grown in a liquid medium made by extraction of malt or grain. The enzyme was primarily used within the textile industry for the removal of the starch that protects the warp in the manufacture of cotton.

In around 1930 it was found that fungal pectinases could be used in the preparation of fruit products. In subsequent years, several other hydrolases were developed and sold commercially (e.g. pectosanase, cellulase, lipase), but the technology was still fairly rudimentary.

After World War Two the fermentation industry underwent rapid development as methods for the production of antibiotics were developed. These methods were soon adapted for the production of enzymes. In the 1960s, glucoamylase was introduced as a means of hydrolysing starch, replacing acid hydrolysis. Subsequently, in the 1960s and 1970s, proteases were incorporated into detergents and then glucose isomerase was introduced to produce sweetening agents in the form of high-fructose syrups. Since the 1990s, lipases have been incorporated into washing powders, and a variety of immobilized enzyme processes have been developed (see section on enzyme immobilization), many of which utilize intracellular enzymes.

Currently, enzymes are used in four distinct fields of commerce and technology ( Table 6 ):

• • as industrial catalysts
• • as therapeutic agents
• • as analytic reagents
• • as manipulative tools (e.g. in genetics).

Of the thousands of different types of enzymes, about 95% are available from suppliers in quantities ranging from μg to kg, provided essentially for research purposes. Around 40–50 enzymes are produced on an industrial scale (i.e. ranging from multiple kilograms to tonnes per annum). The global enzyme market is currently dominated by the hydrolases, especially the proteases, together with amylases, cellulases and lipases supplied either as liquid concentrates or as powders or granules that release the soluble enzyme on dissolution. Global production is dominated by two companies, which between them supply more than two-thirds of the global enzyme market, namely the Danish company Novozymes, with a market share of 47%, and the U.S. company DuPont (which recently acquired Genencor), with 21%.

The value of the world enzyme market has increased steadily from £110 million in 1960 to £200 million in 1970, £270 million in 1980, £1 000 million in 1990 and over £2 000 million in 2010. Food and beverage enzymes represented the largest sector of the industrial enzymes market in 2010, with a value of £750 million, and the market for enzymes for technical applications (including diagnostic applications, research and biotechnology) accounted for a further £700 million. Estimates of future demand are in the range of £4 000–5 000 million between 2015 and 2016, growing at a rate of 6–7% annually. The developing economies of the Asia-Pacific Region, the Middle East and Africa are now seen to be emerging as the fastest growing markets for industrial enzymes.

Microbial enzymes are typically produced in batches by culturing the producing organism within a batch fermenter. Fermentation typically lasts between 30 and 150 h, with the optimum enzyme yield for the process falling somewhere between the optimum biomass yield and the point of maximal enzyme activity within the cells. Relatively small fermenters with a volume of 10–100 m 3 are generally employed, allowing flexibility where a number of different products are being produced. Many production systems are optimized by means of a fed-batch process, in which substrates are gradually fed into the reactor over the course of the fermentation, rather than being provided all at once at the start of the process. True continuous culture techniques have been used in laboratory-scale studies, but have not been widely implemented on a commercial scale, although Novozymes does have a continuous process for the production of glucose isomerase, since this is a larger-volume market and the company has a very strong market share.

Enzyme immobilization

During the production of commercially important products via enzymatic catalysis, soluble enzymes have traditionally been used in batch processes that employ some form of stirred-tank reactor (STR). In these processes, at the end of the batch run the product must be separated from any unused substrate, and also from the enzyme catalyst. Removal of the enzyme at this stage can be achieved by thermal denaturation (only if the product is thermostable) or by ammonium sulfate precipitation or ultrafiltration. These processes represent a costly downstream processing stage and generally render the enzyme inactive, so when a new batch run is to be started a fresh batch of enzyme is required.

Immobilized enzyme systems, in contrast, ‘fix’ the enzyme so that it can be reused many times, which has a significant impact on production costs. As a very simple example, if an enzyme is mixed with a solution of warm (but not too hot) agar and this is allowed to set, the enzyme will be entrapped (for the purposes of this example let us ignore the fact that the enzyme will gradually leak out of this gel). The agar can then be cut up into cubes and these can be placed in a STR, together with substrate, as shown in Figure 12 . Again the reaction would be allowed to proceed (and it might actually be slower due to diffusional constraints and other effects described later). At the end of the batch run the catalyst can now be easily separated from the product by passing the reactor contents through a coarse mesh. Immediately an important downstream processing step has been carried out and, just as importantly, the active enzyme has been recovered so that it can be reused for the next batch run. This ease of separation of enzyme from product is a major advantage of all immobilized systems over their counterparts that use free (i.e. soluble) enzyme.

This physical advantage of ease of reuse of immobilized biocatalysts is one of the main reasons why such systems are favoured commercially. However, immobilization may also produce biochemical changes that lead to enhanced biocatalyst stability, which may be manifested as:

• • an increased rate of catalysis
• • prolonged duration of catalysis
• • greater operational stability to extremes of pH, temperature, etc.

The particular advantage(s) conferred by immobilization will therefore differ from one system to another. It should be noted that often there may be no biochemical advantage at all, and the simple physical advantage of ease of separation of the biocatalyst from the product may be sufficient to favour the commercial development of an immobilized process.

At this point one problem that will immediately spring to mind for most students is that they have always been taught to fully mix all of the reagents of a reaction, yet the basic principle of immobilization is to partition the biocatalyst into a distinct phase, rather than mix it homogeneously with the substrate. Will this not cause reaction rates to be low? The answer to this question is yes, and the relationship between the activity of an immobilized system and a non-immobilized system can be expressed as the effectiveness factor (η), where:

Thus an immobilized system with an effectiveness factor of 0.1 would show only 10% of the activity of a non-immobilized system with the same amount of enzyme and operating under the same conditions. At first sight this might appear to be a major problem. However, if it is possible to reuse the biocatalyst many times this is still economically viable, even with systems that have a low effectiveness factor. In principle, therefore, for economic viability:

Thus if an immobilized system has an effectiveness factor of 0.1 (i.e. 10%) and we can reuse the biocatalyst 10 times, we essentially achieve the same overall catalytic activity with both the non-immobilized system and the immobilized one. However, if we are able to reuse the biocatalyst 100 times we in fact obtain 10 times more total activity from the immobilized system than from the equivalent non-immobilized system, so the immobilized system may be economically preferable.

Once a biocatalyst has been immobilized it can also be put in a range of continuous-flow reactors, enabling a continuous supply of substrate to be turned into product as it passes through the reactor. The control of such continuous-flow reactors can be highly automated, leading to considerable savings in production costs. For example, a STR can be easily modified to produce a continuous-flow stirred-tank reactor (CSTR) ( Figure 13 a), in which the enzyme is held within the reactor by a coarse mesh, and the product continuously flows out of the reactor as substrate is pumped in. It is also possible to produce a packed-bed reactor (PBR) ( Figure 13 b), in which the agar cubes are packed into a column and the substrate is pumped through the bed without any need for stirring.

CSTRs and PBRs enable the enzyme to be reused many times before it needs to be replaced. For example, in the production of high-fructose syrups, the immobilized glucose isomerase enzyme would typically be used continuously for between 2 and 4 months, and only after this time (when its activity would have dropped to 25% of the original level) would it need to be replaced.

The overall operating costs of continuous-flow reactors are often significantly lower than those of equivalent batch processes. Batch reactors need to be emptied and refilled frequently at regular intervals. Not only is this procedure expensive, but it also means that there are considerable periods of time when such reactors are not productive (so-called ‘downtime’). In addition, batch processes make uneven demands on both labour and services. They may also result in pronounced batch-to-batch variations, as the reaction conditions change with time, and they may be difficult to scale up, due to the changing power requirements for efficient mixing. Due to their higher overall process efficiency, continuous processes using immobilized enzymes may be undertaken in production facilities that are around 10 to 100 times smaller than those required for equivalent batch processes using soluble enzymes. Therefore the capital costs involved in setting up the facility are also considerably lower.

Immobilization techniques

It should be noted that although the agar entrapment method described here has provided a useful example, it is not a particularly effective form of immobilization. The high temperature required to prevent the agar from setting may lead to thermal inactivation of the enzyme, and the agar gel itself is very porous and will allow the enzyme to leak out into the surrounding solution.

There are in fact thousands of different techniques of immobilization, all of which are much more effective than our example. In general these techniques can be classified as belonging to one of three categories ( Figure 14 ):

• • adsorption
• • covalent bonding
• • entrapment.

The physical adsorption of an enzyme to a supporting matrix is the oldest method of immobilization. As early as 1916, J.M. Nelson and Edward G. Griffin described the adsorption of yeast invertase on to activated charcoal, and the subsequent use of this preparation for sucrose hydrolysis. Over the years a variety of adsorbents have been used, including cellulose, Sephadex, polystyrene, kaolinite, collagen, alumina, silica gel and glass. Such immobilization procedures are extremely easy to perform, as the adsorbent and enzyme are simply stirred together for a time (typically minutes to hours). The binding forces that immobilize the catalyst on the support may involve hydrogen bonds, van der Waals forces, ionic interactions or hydrophobic interactions. Such forces are generally weak in comparison with covalent bonds—for example, a hydrogen bond has an energy content of about 20 kJ mol −1 , compared with 200–500 kJ mol −1 for a covalent bond. Thus, when using such methods, yields (i.e. the amount of enzyme bound per unit of adsorbent) are generally low. In addition, adsorption is generally easily reversed, and can lead to desorption of the enzyme at a critical time.

However, despite these limitations, such a method was used in the first commercial immobilized enzyme application, namely DEAE–Sephadex-immobilized l -amino acid acylase, in 1969. DEAE–Sephadex is an ion-exchange resin that consists of an inert dextran particle activated by the addition of numerous diethylaminoethyl groups. Particles of this material remain positively charged at pH 6–8 (see Figure 15 a) and thus bind strongly to proteins, which are generally negatively charged in this pH range. If the pH is kept constant, the enzyme and support will remain ionically linked. However, when over time the enzyme loses its activity through denaturation, the pH can be adjusted to a more acidic value, the old enzyme will be desorbed, and the pH can then be readjusted back to pH 6–8 and a fresh batch of enzyme bound. Thus the support matrix may be used many times, giving the process significant economic benefits.

Clearly DEAE–Sephadex immobilization is only of value for enzymes that have a neutral-to-alkaline pH optimum. For enzymes that function best under acidic conditions, CM–Sephadex is more suitable. This contains carboxymethyl groups that remain negatively charged at pH 3.5–4.5 ( Figure 15 b). Proteins at this pH are generally positively charged and will thus ionically bind to the support. Desorption of the enzyme will occur when the pH is adjusted to a more alkaline value.

Due to the simplicity and controllability of this immobilization procedure, combined with the economic benefits of reuse of the support, ion-exchange materials are now widely used as the method of choice in many industrial settings.

Covalent bonding

Immobilization of enzymes by covalent bonding to activated polymers is a widely used approach since, although it is often a tedious procedure, it is capable of producing an immobilized enzyme that is firmly bound to its support. The range of polymers and chemical coupling procedures that are used is enormous.

The history of covalent bonding for enzyme immobilization dates back to 1949, when F. Michael and J. Ewers used the azide derivative of carboxymethylcellulose to immobilize a variety of proteins. Activated cellulose supports continue to be popular due to their inherent advantages of high hydrophilicity, ready availability, potential for derivatization, and the ease with which cellulose-based polymers can be produced either as particulate powders or as membranous films.

It is often more effective not to build the reactive group into the cellulose itself, but instead to use a chemical ‘bridge’ between the cellulose and the enzyme molecule. The requirements for such a bridging or linking molecule are that it must be small, and that once it has reacted with the support it must have a further reactive group capable of reacting with the enzyme. An example of such a bridging molecule is glutaraldehyde, which contains two aldehyde groups, one at either end of its (CH 2 ) 3 moiety. At neutral pH values the aldehyde groups will react with free amino groups. Thus one end of the glutaraldehyde molecule may be attached to the support, and the other to the enzyme.

Covalently immobilized enzymes are strongly bound to their support, so when the proteins denature they are difficult to remove (in contrast to adsorption, as described earlier). Therefore it is usual for both the enzyme and the support to be replaced. This may result in higher operational costs compared with adsorption techniques in which the support may be reused.

The entrapment of an enzyme can be achieved in a number of ways:

• • inclusion within the matrix of a highly cross-linked polymer
• • separation from the bulk phase by a semi-permeable ‘microcapsule’
• • dissolution in a distinct non-aqueous phase.

An important feature of entrapment techniques is that the enzyme is not in fact attached to anything. Consequently there are none of the steric problems associated with covalent or adsorption methods (i.e. the possibility of the enzyme binding in such a way that its active site is obstructed by part of the supporting polymer matrix).

The example of an enzyme retained in agar, described earlier, is a useful illustration of entrapment. A preferable alternative involves mixing the catalyst with sodium alginate gel and extruding this into a solution of calcium chloride to produce solid calcium alginate particles. This technique has the advantage of not requiring the use of high temperatures. However, although it is a popular activity in teaching laboratories, outside that setting it is generally unsuitable for the immobilization of purified enzymes, as these are often able to leak out of the gel. Entrapment techniques for purified enzymes are more likely to involve retaining the enzyme behind some form of ultrafiltration membrane. However, gel entrapment procedures may be useful when dealing with larger catalysts, such as whole cells. For example, gel-immobilized living yeast cells have been used successfully in the manufacture of champagne by Moët & Chandon.

Immobilization: changes in enzyme properties

Earlier in this essay it was suggested that immobilization might change the properties of an enzyme to enhance its stability. Initially it was believed that such enhanced stability resulted from the formation of bonds between the enzyme and the supporting matrix that physically stabilize the structure of the protein. Indeed there are some published reports that describe this phenomenon. With regard to the stabilization of proteolytic enzymes, which often exhibit more prolonged activity in the immobilized state, this is most probably explained by the fact that such proteases in free solution are prone to autodigestion (i.e. enzyme molecules cleave the peptide bonds of adjacent enzyme molecules), a process that is largely prevented when they are fixed to a supporting matrix.

However, the effects of immobilization are more often due to the supporting matrix changing the microenvironment around the enzyme and/or introducing diffusional constraints that modify the activity of the catalyst. Consider, for example, immobilization of the enzyme by adsorption on to a polyanionic (negatively charged) support such as cellulose. If the substrate is a cation (i.e. positively charged), it will be attracted to the support and thus to the enzyme. In this case the enzyme might well display higher activity, as the substrate concentration in its microenvironment would be higher than that in the surrounding bulk phase. Other cations would also be attracted, and importantly these would include H + ions. Thus the microenvironment would also be enriched in H + ions, so the pH surrounding the enzyme would be lower than the pH of the bulk phase. Consequently the enzyme would also exhibit an altered pH profile compared with that of its soluble counterpart.

In addition, the immobilization matrix might act as a barrier to the diffusion of substrates, products and other molecules. For example, if a high enzyme loading was put into a gel particle and this was then immersed in substrate solution, the substrate would diffuse into the gel and rapidly be converted into product. Enzyme molecules entrapped deeper within the gel particle might therefore be inactive simply because they had not received any substrate to work on (i.e. all of the substrate was converted to product in the outer layers of the particle). Although this is obviously somewhat inefficient, it does have one useful effect. When over time the enzyme within the system denatures, the loss of activity of the enzyme in the outer part of the particle means that substrate will now diffuse deeper into the particle to reach the previously unused core enzyme molecules. In effect this inner reserve of enzyme will offset the loss of enzyme activity through denaturation, so the system will show little or no overall loss of activity. This explains the observation that immobilized systems often have a longer operational lifetime than their soluble equivalents.

In addition, it is of interest that enzymes bound to natural cell membranes (phospholipid bilayers) within living cells will also probably demonstrate these effects, and immobilized systems thus provide useful models for the study of such membrane-bound proteins in living cells.

Immobilized enzymes at work

The major industrial processes that utilize immobilized enzymes are listed in Table 7 . Sales of immobilized enzymes peaked in 1990, when they accounted for about 20% of all industrial enzyme sales, almost entirely due to the use of glucose isomerase for the production of sweetening agents. Other commercial applications utilize penicillin acylase, fumarase, β–galactosidase and amino acid acylase. Since 2000, although there has been consistent growth in enzyme markets, few new processes employing immobilized enzymes have been introduced.

The following three examples highlight many of the biochemical, technological and economic considerations relating to the use of immobilized enzymes on a commercial and industrial scale.

Production of high-fructose syrup

Undoubtedly the most significant large-scale application of immobilized enzymes involves the production of high-fructose corn syrup (HFCS). Although most of the general public believe that sucrose is responsible for the ‘sweetness’ of food and drinks, there have been significant efforts to replace sucrose with alternative, and often cheaper, soluble caloric sweetening agents. HFCS is a soluble sweetener that has been used in many carbonated soft drinks since the 1980s, including brand-name colas such as Coca-Cola and Pepsi-Cola. HFCS is produced by the enzymatic digestion of starch derived from corn (maize). Developments in HFCS production have been most prominent in countries such as the U.S.A., which have a high capacity to produce starch in the form of corn, but which do not cultivate significant amounts of sugar cane or sugar beet, and must therefore import either the raw products (for processing) or the refined sugar (sucrose) itself.

Simple corn syrups can be manufactured by breaking down starch derived from corn using the enzyme glucoamylase alone or in combination with α-amylase. These enzymes are cheap and can be used in a soluble form. Since starch has to be extracted from corn at high temperatures (because starch has poor solubility at low temperatures and forms very viscous solutions), the process utilizes enzymes from thermophilic organisms, which have very high temperature optima. Simple corn syrup is therefore composed predominantly of glucose, which unfortunately has only 75% of the sweetness of sucrose. However, in order to make the syrup sweeter the enzyme glucose isomerase, which catalyses the following reaction, can be employed:

This enzyme (described previously in the section on properties and mechanisms of enzyme action) will produce a roughly 50:50 mixture of glucose and fructose at equilibrium, and since fructose has 150% of the sweetness of sucrose, this glucose:fructose mixture will have a similar level of sweetness to sucrose. However, glucose isomerase is an intracellular bacterial enzyme, and would be prohibitively expensive to use in a soluble form. This makes it an ideal candidate for use in an immobilized process.

The first glucose isomerase enzyme to be isolated was obtained from species of Pseudomona s in 1957, and more useful enzymes were isolated throughout the 1960s from species of Bacillus and Streptomyces. In 1967, the Clinton Corn Processing Company of Iowa, U.S.A. (later renamed CPC International) introduced a batch process that utilized an immobilized glucose isomerase enzyme, and by 1972 the company had developed a continuous process for the manufacture of HFCS containing 42% fructose using a glucose isomerase enzyme immobilized on a DEAE ion-exchange support.

During the late 1970s, advances in enzymology, process engineering and fractionation technology led to the production of syrups with a higher fructose content, and today HFCS containing 55% fructose is generally produced, and is commonly used in soft drinks, although 42% fructose syrups are still also produced for use in some processes, including the production of bakery foodstuffs.

In 2010, the U.S. production of HFCS was approximately 8 million metric tons, accounting for 37% of the U.S. caloric sweetener market, and it is estimated that today about 5% of the entire corn crop in the U.S.A. is used to produce HFCS.

Hydrolysis of lactose

Within the dairy industry the production of 1 kg of cheese requires about 10 litres of milk, and produces about 9 litres of whey as a waste product. Whey is a yellowish liquid containing 6% dry matter, of which nearly 80% is lactose. The enzyme lactase (β-galactosidase) may be used to break down lactose to its constituent monosaccharides, namely glucose and galactose, which are more soluble than lactose, and have potential uses as carbon sources in microbial fermentation, and can also be used as caloric sweeteners.

Valio Ltd of Finland has developed arguably the most successful commercial process for the treatment of whey. Using a lactase enzyme obtained from Aspergillus , immobilized by adsorption and cross-linked on to a support resin, whey syrups are produced that have been utilized as an ingredient in drinks, ice cream and confectionery products. The Aspergillus enzyme has an acid pH optimum of 3–5, and by operating at low pH the process avoids excessive microbial contamination. Treatment plants that utilize 600-litre columns have been built in Finland, and these are used to treat 80 000 litres of whey per day. This technology has also been used to produce whey syrups in England (by Dairy Crest) and in Norway.

Similar technology can also be used to remove lactose from milk. Lactose-free milk is produced for consumption by those who have lactose intolerance (a genetic condition), and also for consumption by pets such as cats, which are often unable to digest lactose easily. The first industrial processing facility to use immobilized lactase to treat milk was opened in 1975, when Centrale del Latte of Milan, Italy, utilized a batch process in which yeast ( Saccharomyces ) lactase, with a neutral pH optimum of 6–8, was immobilized within hollow permeable fibres. This process was capable of treating 10 000 litres of milk per day, and was operated at low temperature to prevent microbial contamination.

Production of semi-synthetic penicillins

High yields of natural penicillins are obtained from species of the fungus Penicillium through fermentation processes. However, over the years many microbial pathogens have become resistant to natural penicillins, and are now only treatable with semi-synthetic derivatives. These are produced through cleavage of natural penicillin, such that the G or V side chain is removed from the 6-aminopenicillanic acid (6-APA) nucleus of the molecule:

Thereafter, by attachment of a chemically different side chain, a semi-synthetic penicillin product (e.g. ampicillin, amoxicillin) can be formed. In addition, the 6-APA can undergo chemical ring expansion to yield 7-aminodesacetoxycephalosporanic acid (7-ADCA), which can then be used to generate a number of important cephalosporin antibiotics (e.g. cephalexin, cephradine, cefadroxil).

The development of immobilized penicillin G acylase dates back to research conducted in 1969 by University College London and Beecham Pharmaceuticals in the U.K. Penicillin G acylases are intracellular enzymes found in E. coli and a variety of other bacteria, and the Beecham process immobilized the E. coli enzyme on a DEAE ion-exchange support. Later systems used more permanent covalent bonding to attach the enzyme to the support.

In the 1980s and 1990s, world production of penicillins was dominated by European manufacturers, which accounted for production of around 30 000 tonnes of penicillin per annum, 75% of which was used for the manufacture of semi-synthetic penicillins and cephalosporins. However, over the past 10 years, due to increasing costs of labour, energy and raw materials, more bulk manufacturing has moved to the Far East, where China, Korea and India have become major producers. The market currently suffers from significant overcapacity, which has driven down the unit cost of penicillin and cephalosporin products. However, penicillins and cephalosporins still represent one of the world's major biotechnology markets, with annual sales of about £10 000 million, accounting for 65% of the entire global antibiotics market.

Enzymes in analysis

Enzymes have a wide variety of uses in analytical procedures. Their specificity and potency allow both detection and amplification of a target analyte. ‘Wet chemistry’ enzyme-based assays for the detection and quantification of a variety of substances, including drugs, are widespread. Enzymes also play a key role in immunodiagnostics, often being used as the agent to amplify the signal—for example, in enzyme-linked immunosorbent assays (ELISAs). Within DNA-fingerprinting technology, the enzyme DNA polymerase plays a key role in the amplification of DNA molecules in the polymerase chain reaction. However, ‘wet chemistry’ analytical methods are increasingly being replaced by the use of biosensors—that is, self-contained integrated devices which incorporate a biological recognition component (usually an immobilized enzyme) and an electrochemical detector (known as a transducer).

Much of the technological development of biosensors has been motivated by the need to measure blood glucose levels. In 2000, the World Health Organization estimated that over 170 million people had diabetes, and predicted that this figure will rise to over 360 million by 2030. In view of this, many companies have made significant investments in R&D programmes that have led to the availability of a wide variety of glucose biosensor devices.

In 1962, Leland Clark Jr coined the term ‘enzyme electrode’ to describe a device in which a traditional electrode could be modified to respond to other materials by the inclusion of a nearby enzyme layer. Clark's ideas became a commercial reality in 1975 with the successful launch of the Yellow Springs Instruments (YSI) model 23A glucose analyser. This device incorporated glucose oxidase together with a peroxide-sensitive electrode to measure the hydrogen peroxide (H 2 O 2 ) produced during the following reaction:

In this device, the rate of H 2 O 2 formation is a measure of the rate of the reaction, which depends on the concentration of glucose in solution, thus allowing the latter to be estimated.

As was discussed earlier, in enzyme-catalysed reactions the relationship between substrate concentration and reaction rate is not linear, but hyperbolic (as described by the Michaelis–Menten equation). This is also true for the glucose oxidase within a biosensor. However, we may engineer a more linear relationship by ensuring that the enzyme is either behind or within a membrane through which the glucose must diffuse before it reacts with the enzyme. This means that the system becomes diffusionally, rather than kinetically, limited, and the response is then more linearly related to the concentration of glucose in solution.

Over the years the YSI model 23A glucose analyser has been replaced by a range of much more advanced models. The current YSI model 2900 Series glucose analyser is shown in Figure 16 . This instrument has a 96-sample rack that enables batches of samples to be run, with the analysis of each sample taking less than a minute. The instrument can measure the glucose content of whole blood, plasma or serum, and requires only 10 μl of sample per analysis. The membrane-bound glucose oxidase typically only needs to be replaced every 3 weeks, thereby reducing the cost of analysis. These systems also offer advanced data-handling and data-storage facilities.

In addition, these instruments can be modified to analyse a wide variety of other substances of biological interest, simply by incorporating other oxidase enzymes into the membrane ( Table 8 ).

To enable diabetic patients to take their own blood glucose measurements, small hand-held biosensors have also been developed, which are in fact technologically more advanced because the enzyme and transducer are more intimately linked on the sensor surface. The first device of this type was launched in 1986 by Medisense, and was based on technology developed in the U.K. at Cranfield and Oxford Universities. The ExacTech blood glucose meter was the size and shape of a pen, and used disposable electrode strips. This device was followed by a credit card-style meter in 1989. Such devices again rely on glucose oxidase as the biological component, but do not measure the reaction rate via the production (and detection) of H 2 O 2 . Instead they rely on direct measurement of the rate of electron flow from glucose to the electrode surface. The reactions that occur within this device may be summarized as follows:

and at the electrode surface:

where GO x -FAD represents the FAD redox centre of glucose oxidase in its oxidized form, and GO x -FADH 2 represents the reduced form.

Basically electrons are removed from the glucose molecules and passed via the enzyme to the ferrocene mediator, which then donates them to the working electrode surface, resulting in the generation of an electrical current that is directly proportional to the rate of oxidation of glucose, and thus proportional to the glucose concentration in the sample.

Medisense, whose only product was its blood glucose meter, was bought by Abbott Diagnostics in 1996, and Abbott-branded devices continued to use and develop this technology for some time.

In 1999, Therasense marketed a glucose meter that represented the next generation of sensing technology, and integrated the enzyme even more closely with the electrode. Originally developed by Adam Heller at the University of Texas in the 1990s, wired-enzyme electrodes do not rely on a soluble mediator such as the ferrocene used in the Medisense devices. Instead the enzyme is immobilized in an osmium-based polyvinyl imidazole hydrogel in which the electrons are passed from enzyme to electrode by a series of fixed electroactive osmium centres that shuttle the electrons onward in a process called ‘electron hopping.’

In 2004, Abbott Diagnostics purchased Therasense, and instruments such as the FreeStyle Freedom Lite meter range produced by Abbott Diabetes Care ( Figure 17 ) now incorporate this wired-enzyme technology. Devices of this type are highly amenable to miniaturization.

Continuous measuring devices are becoming increasingly available, and may well revolutionize the control of certain disease conditions. For example, with regard to diabetes, devices such as the FreeStyle Navigator range from Abbott Diabetes Care use the same wired-enzyme technology as that described earlier, but now incorporate this into a tiny filament about the diameter of a thin hypodermic needle. This is inserted approximately 5 mm under the skin to measure the glucose level in the interstitial fluid that flows between the cells. The unit is designed to remain in situ for up to 5 days, during which time it can measure the glucose concentration every minute. A wireless transmitter sends the glucose readings to a separate receiver anywhere within a 30-metre range, and this can then issue an early warning alarm to alert the user to a falling or rising glucose level in time for them to take appropriate action and avoid a hypoglycaemic or hyperglycaemic episode.

In addition, experimental units have already been developed that link continuous glucose biosensor measurement systems with pumps capable of gradually dispensing insulin such that the diabetic condition is automatically and reliably controlled, thereby avoiding the traditional peaks and troughs in glucose levels that occur with conventional glucose measurement and the intermittent administration of insulin.

Therefore, looking to the future, we may confidently expect to see the development of biosensor systems that can continuously monitor a range of physiologically important analytes and automatically dispense the required medication to alleviate the symptoms of a number of long-term chronic human illnesses.

Closing remarks

For the sake of conciseness, this guide has been limited to some of the basic principles of enzymology, together with an overview of the biotechnological applications of enzymes. It is important to understand the relationship between proteins and the nucleic acids (DNA and RNA) that provide the blueprint for the assembly of proteins within the cell. Genetic engineering is thus predominantly concerned with modifying the proteins that a cell contains, and genetic defects (in medicine) generally relate to the abnormalities that occur in the proteins within cells. Much of the molecular age of biochemistry is therefore very much focused on the study of the cell, its enzymes and other proteins, and their functions.

Abbreviations

This article is a reviewed, revised and updated version of the following ‘Biochemistry Across the School Curriculum’ (BASC) booklet: Teal A.R. & Wymer P.E.O., 1995: Enzymes and their Role in Technology. For further information and to provide feedback on this or any other Biochemical Society education resource, please contact gro.yrtsimehcoib@noitacude . For further information on other Biochemical Society publications, please visit www.biochemistry.org/publications .

Recommended reading and key publications

1. historically important landmark papers (in chronological order).

• Takamine J. Process of making diastatic enzyme. 1894 U.S. Pat. 525,823. Describes the first commercial exploitation of semi-purified enzymes in the West. [ Google Scholar ]
• Briggs G.E., Haldane J.B.S. A note on the kinetics of enzyme action. Biochem. J. 1925; 19 :338–339. A classic paper in which the steady-state assumption was introduced into the derivation of the Michaelis–Menten equation. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Koshland D.E., Jr Application of a theory of enzyme specificity to protein synthesis. Proc. Natl Acad. Sci. U.S.A. 1958; 44 :98–104. Describes the proposal of an ‘induced fit’ mechanism of substrate binding. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Clark L.C., Jr, Lyons C. Electrode systems for continuous monitoring in cardiovascular surgery. Ann. N.Y. Acad. Sci. 1962; 102 :29–45. Introduces the concept of a biosensor for measuring blood glucose levels during surgery. [ PubMed ] [ Google Scholar ]
• Monod J., Wyman H., Changeux J.P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 1965; 12 :88–118. Describes the ‘concerted’ model of transitions of allosteric proteins in which all constituent monomers are in either the T-state or the R-state. [ PubMed ] [ Google Scholar ]
• Koshland D.E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966; 5 :365–385. Describes the ‘sequential’ model of transitions of allosteric proteins in which protein moves through hybrid structures with some monomers in the T-state and some in the R-state. [ PubMed ] [ Google Scholar ]
• Updike S.J., Hicks G.P. The enzyme electrode. Nature. 1967; 214 :986–988. Describes the simplification of the electrochemical assay of glucose by immobilizing and thereby stabilizing the glucose oxidase enzyme. [ PubMed ] [ Google Scholar ]
• Tramontano A., Janda K.D., Lerner R.A. Catalytic antibodies. Science. 1986; 234 :1566–1570. [ PubMed ] [ Google Scholar ]
• Pollack S.J., Jacobs J.W., Schultz P.G. Selective chemical catalysis by an antibody. Science. 1986; 234 :1570–1573. The first reports of antibody proteins that demonstrate catalytic activity. [ PubMed ] [ Google Scholar ]
• Johnson K.A., Goody R.S. The original Michaelis constant: translation of the 1913 Michaelis–Menten paper. Biochemistry. 2011; 50 :8264–8269. A modern translation, commentary and re-analysis of the original 1913 paper, Die Kinetik der Invertinwirkung. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Taylor A.I., Pinheiro V.B., Smola M.J., Morgunov A.S., Peak-Chew S., Cozens C., Weeks K.M., Herdewijn P., Holliger P. Catalysts from synthetic genetic polymers. Nature. 2015; 518 :427–430. Describes the first artificial enzymes to be created using synthetic biology. [ PMC free article ] [ PubMed ] [ Google Scholar ]

2. Enzyme principles

• Changeux J.-P. 50 years of allosteric interactions: the twists and turns of the models. Nat. Rev. Mol. Cell Biol. 2013; 14 :819–829. [ PubMed ] [ Google Scholar ]
• Kamata K., Mitsuya M., Nishimura T., Eiki J., Nagata Y. Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase. Structure. 2004; 12 :429–438. [ PubMed ] [ Google Scholar ]

3. Enzyme applications

• Adrio J.L., Demain A.L. Microbial enzymes: tools for biotechnological processes. Biomolecules. 2014; 4 :117–139. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Clarke S.F., Foster J.R. A history of blood glucose meters and their role in self-monitoring of diabetes mellitus. Br. J. Biomed. Sci. 2012; 69 :83–93. [ PubMed ] [ Google Scholar ]
• Fernandes P. Enzymes in food processing: a condensed overview on strategies for better biocatalysts. Enzyme Res. 2010; 2010 :862537. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Vashist S.K., Zheng D., Al-Rubeaan K., Luong J.H.T., Sheu F.-S. Technology behind commercial devices for blood glucose monitoring in diabetes management: a review. Anal. Chim. Acta. 2011; 703 :124–136. [ PubMed ] [ Google Scholar ]
• Vellard M. The enzyme as drug: application of enzymes as pharmaceuticals. Curr. Opin. Biotechnol. 2003; 14 :444–450. [ PubMed ] [ Google Scholar ]
• Woodley J.M. New opportunities for biocatalysis: making pharmaceutical processes greener. Trends Biotechnol. 2008; 26 :321–327. [ PubMed ] [ Google Scholar ]

4. Useful textbooks

• Bisswanger H. Enzyme Kinetics: Principles and Methods. 2nd edn. Weinheim, Germany: Wiley-VCH; 2008. Available online and as hard copy. A user-friendly and comprehensive treatise on enzyme kinetics. [ Google Scholar ]
• Buchholz K., Kasche V., Bornscheuer U.T. Biocatalysts and Enzyme Technology. 2nd edn. Weinheim, Germany: Wiley-VCH; 2012. Best-selling textbook that provides an instructive and comprehensive overview of our current knowledge of biocatalysis and enzyme technology. [ Google Scholar ]
• Copeland R.A. In: Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists. 2nd edn. Hoboken NJ., editor. John Wiley & Sons, Inc; 2013. Provides thorough coverage of both the principles and applications of enzyme inhibitors. [ PubMed ] [ Google Scholar ]
• McGrath M.J., Scanaill C.N. Sensor Technologies: Healthcare, Wellness and Environmental Applications. New York: Apress Media, LLC; 2014. Available online. Covers sensor technologies and their clinical applications, together with broader applications that are relevant to wellness, fitness, lifestyle and the environment. [ Google Scholar ]
• Trevan M.D. Immobilized Enzymes: An Introduction and Applications in Biotechnology. Chichester: John Wiley & Sons; 1980. An older text, and difficult to find except in libraries, but it provides an introductory text for non-experts, and as yet there is no other book that fulfils this role. [ Google Scholar ]
• Whitehurst R.J., van Oort M. Enzymes in Food Technology. 2nd edn. Chichester: Wiley-Blackwell; 2009. Provides comprehensive coverage of the widespread use of enzymes in food-processing improvement and innovation. [ Google Scholar ]

Enzymes: Structure, Functions, and Classification

Enzymes are biological catalysts with extraordinary catalytic power. They are central to every biochemical process.  

Chemical reactions like sucrose oxidation cannot happen in the correct time frame and thus cannot sustain life. Hence, as biological catalysts, enzymes play a significant role in biochemical processes. 

Table of Contents

General Properties of Enzymes

Structure of enzymes, primary structure, secondary structure.

Enzymes often exhibit secondary structures such as alpha-helices and beta-sheets. These structures result from hydrogen bonding between amino acids in the polypeptide chain. The secondary structure helps determine the overall folding of the enzyme.

Tertiary Structure

It is the three-dimensional arrangement of the entire polypeptide chain, including the secondary structural elements. This structure is stabilized by various interactions such as hydrogen bonds, disulfide bonds, hydrophobic interactions, and electrostatic interactions between amino acid side chains.

Quaternary Structure (if applicable)

Active site, regulatory sites (if applicable).

Some enzymes have regulatory sites separate from the active site, where molecules such as inhibitors or activators can bind. These regulatory molecules can modulate the enzyme’s activity, inhibiting or enhancing its catalytic function.

Other structural components of enzymes

Classification and nomenclature of enzymes.

The naming of many enzymes is done by adding the suffix “-ase” to their substrates’ name or a word or phrase that describes their activity. Thus, DNA polymerase catalyzes the polymerization of nucleotides to form DNA, and urease catalyzes the hydrolysis of urea. Before the specific reaction catalyzed was known, other enzymes were named for a broad function. For example, pepsin, from the Greek word pepsis, meaning “digestion,” was given to an enzyme known to act in the digestion of foods. 

Note: The function of most enzymes is the catalysis of the transfer of atoms, electrons, or functional groups. Therefore, they have different classifications, code numbers, and names assigned according to the type of transfer reaction, the donor group, and the group acceptor.

For example, the formal systematic name of the enzyme catalyzing the reaction, ATP + D-glucose → ADP + D-glucose 6-phosphate, is ATP: glucose phosphotransferase. This denotes that the enzyme is the catalyst for transferring a phosphoryl group from ATP to glucose. 

How do Enzymes Work?

Substrate binding.

The enzyme’s active site, which has a specific three-dimensional shape complementary to the substrate molecule, initially binds to the substrate. This binding can occur through a lock-and-key mechanism (where the substrate fits precisely into the active site) or an induced fit mechanism (where the active site reshapes slightly to accommodate the substrate).

Formation of the Enzyme-Substrate Complex

Active site residues  , transition state stabilization.

Enzymes stabilize the reaction’s transition state, the high-energy intermediate state that occurs during the conversion of substrate into product. By stabilizing the transition state, enzymes lower the activation barrier, making it easier for the reaction to proceed.

Product Release

Enzyme-product complex, factors affecting enzyme activity, temperature.

Enzyme activity is susceptible to temperature changes. Generally, increasing temperature increases the rate of enzymatic reactions by providing more kinetic energy to molecules, which enhances their collision frequency and leads to more successful enzyme-substrate interactions. However, extremely high temperatures can denature enzymes, causing loss of their catalytic activity due to disruption of their three-dimensional structure. Each enzyme can operate at an optimal temperature range with maximum activity, known as the temperature optimum.

Substrate Concentration 

Enzyme concentration, cofactors and coenzymes.

Enzyme activity can be inhibited by various molecules known as inhibitors. Inhibitors are of two types: irreversible and reversible inhibitors. Reversible inhibitors include competitive inhibitors (compete with the substrate for the active site), non-competitive inhibitors (bind to an allosteric site and change the enzyme’s conformation), and uncompetitive inhibitors (bind to the enzyme-substrate complex). Irreversible inhibitors covalently bind to the enzyme, permanently inhibiting its activity.

Activators and Modulators

Enzyme structure and conformational changes, functions of enzymes.

These play a crucial role in digestion by breaking down complex macromolecules into smaller, absorbable molecules. For example:

Energy Production

Synthesis of biomolecules, detoxification, cell signaling, immune response.

Enzymes are part of the immune system’s defense mechanisms. For instance, enzymes like lysozyme and proteases in tears, saliva, and mucus help protect against pathogens by breaking down their cell walls or proteins.

Repair and Maintenance

Regulation of metabolic pathways.

Hello, I am Ashma Shrestha. I had recently completed my Masters degree in Medical Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology.

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Recent Posts

Understanding the One Gene One Enzyme Hypothesis – Unlocking the Secrets of Protein Synthesis

• Post author By admin-science
• Post date 20.12.2023

The one gene one enzyme hypothesis is a theory that was proposed by the Nobel Prize-winning scientist George Beadle and Edward Tatum. This hypothesis states that each gene in an organism is responsible for producing one specific enzyme.

An enzyme is a type of protein that plays a vital role in various biochemical reactions within the body. These reactions are essential for the functioning and survival of an organism. Enzymes act as catalysts, speeding up chemical reactions and allowing them to occur more quickly and efficiently.

The one gene one enzyme hypothesis suggests that the genes in an organism are responsible for producing the specific enzymes needed for various biochemical reactions. Each gene codes for the production of a particular enzyme, and any mutations or changes in the gene sequence can lead to the production of a non-functional enzyme or the absence of a necessary enzyme.

This hypothesis revolutionized the field of genetics and provided a framework for understanding how genes and enzymes play a crucial role in the structure and function of living organisms. It laid the foundation for the study of genetic disorders caused by enzyme deficiencies and has contributed to our understanding of various metabolic pathways.

The origins of the one gene one enzyme hypothesis

The one gene one enzyme hypothesis is a fundamental concept in molecular biology, which proposes that each gene controls the production of a specific enzyme. This hypothesis was first proposed by American geneticist George W. Beadle and Edward L. Tatum in the early 1940s.

At the time, the role of genes in controlling biochemical reactions was not well understood. However, Beadle and Tatum’s experiments with the bread mold Neurospora crassa provided crucial evidence to support this hypothesis.

In their experiments, Beadle and Tatum exposed Neurospora crassa to radiation, causing mutations in its genetic material. They then observed the effects of these mutations on the ability of the mold to grow on different types of media.

They found that each mutation affected the mold’s ability to produce a specific enzyme necessary for the breakdown of certain molecules. These observations led them to conclude that each gene controls the production of a specific enzyme.

Based on their findings, Beadle and Tatum proposed the one gene one enzyme hypothesis in 1941. This hypothesis revolutionized our understanding of genetics and laid the foundation for modern molecular biology.

Since then, the one gene one enzyme hypothesis has been refined and expanded upon. It is now recognized that genes can control the production of multiple enzymes through complex regulatory mechanisms. However, the basic principle that each gene codes for a specific enzyme remains valid.

Today, the one gene one enzyme hypothesis is a cornerstone of molecular genetics, providing a fundamental framework for studying the relationship between genes and proteins in various biological processes.

The basic principles of the one gene one enzyme hypothesis

The one gene one enzyme hypothesis is a fundamental concept in genetics that proposes that each gene is responsible for the production of a specific enzyme. This hypothesis was first proposed by George Beadle and Edward Tatum in the early 1940s, based on their experiments with the bread mold Neurospora crassa.

The hypothesis suggests that genes are the instructions for the production of enzymes, which are molecules that catalyze specific chemical reactions within cells. Each gene controls the production of one specific enzyme, and therefore, the phenotype of an organism is determined by the presence or absence of specific enzymes.

According to this hypothesis, mutations in genes can lead to the production of faulty enzymes or the absence of certain enzymes, resulting in metabolic disorders and genetic diseases. For example, the genetic disorder phenylketonuria is caused by a mutation in the gene that codes for the enzyme phenylalanine hydroxylase, which is essential for the breakdown of the amino acid phenylalanine.

The one gene one enzyme hypothesis revolutionized our understanding of the relationship between genes and enzymes. It provided a foundation for the field of molecular genetics and paved the way for advances in genetic engineering and biotechnology.

The role of genes in enzyme production

Genes play a crucial role in the production of enzymes. Enzymes are proteins that act as catalysts in various biochemical reactions, helping to speed up or facilitate these reactions. Each enzyme corresponds to a specific gene, and the production of enzymes is regulated by the expression of their corresponding genes.

What is the one gene one enzyme hypothesis?

The one gene one enzyme hypothesis, proposed by American geneticist George Beadle in the 1940s, states that a single gene is responsible for the synthesis of a specific enzyme. This hypothesis was based on Beadle’s experiments with the bread mold Neurospora crassa, where he observed that mutations in specific genes resulted in the loss of specific enzyme activities.

This hypothesis provided a foundation for understanding the relationship between genes and enzymes. It suggested that genes contain the instructions necessary for the synthesis of enzymes and that mutations in genes can lead to defects in enzyme production, which can have significant physiological effects.

Further research has expanded our understanding of the gene-enzyme relationship. We now know that genes code for the production of not only enzymes but also other functional proteins. Additionally, the one gene one enzyme hypothesis has been refined to the concept of one gene one polypeptide, as genes can code for the synthesis of polypeptides that are not enzymatic in nature.

The impact of the one gene one enzyme hypothesis on genetics

The one gene one enzyme hypothesis is a fundamental concept in genetics that postulates that each gene is responsible for the production of a specific enzyme. This hypothesis was proposed by George Beadle and Edward Tatum in the early 1940s based on their experiments with bread mold.

Before the one gene one enzyme hypothesis, it was not clear how genetic information was related to the phenotype of an organism. This hypothesis provided a crucial link between genes and enzymes, allowing researchers to understand how genetic mutations could lead to biochemical defects.

Understanding enzyme function

Enzymes are proteins that act as catalysts for biochemical reactions in living organisms. They play a vital role in various metabolic processes, such as the breakdown of food molecules and the synthesis of essential molecules. The one gene one enzyme hypothesis provided insight into how enzymes are produced and how their activity is regulated.

According to this hypothesis, each gene encodes the information for the production of a specific enzyme. A mutation in a gene could lead to the production of a dysfunctional enzyme or the complete absence of an enzyme, resulting in a biochemical defect. This knowledge has been invaluable in understanding the genetic basis of many diseases and disorders.

Advancing genetics research

The one gene one enzyme hypothesis revolutionized genetics research by providing a framework for studying the relationship between genes, enzymes, and phenotypes. It allowed researchers to conduct experiments to determine the specific genes responsible for various enzyme activities and their associated phenotypic traits.

This hypothesis also paved the way for the development of new techniques, such as gene knockout and gene editing, which have become indispensable tools in genetic research. By targeting specific genes and altering their function, researchers can now investigate the role of individual genes and enzymes in biological processes.

In conclusion, the one gene one enzyme hypothesis has had a profound impact on genetics. It has provided a key understanding of the relationship between genes, enzymes, and phenotypes, leading to significant advancements in genetic research and our understanding of various diseases.

The discovery of the one gene one enzyme hypothesis

In the field of genetics, the discovery of the one gene one enzyme hypothesis was a groundbreaking milestone that revolutionized our understanding of how genes and enzymes are related. This hypothesis states that each gene is responsible for the production of a specific enzyme.

The initial experiments that led to this hypothesis were conducted by the scientists George Beadle and Edward Tatum in the early 1940s. They studied a fungus called Neurospora crassa and observed that mutations in specific genes resulted in the loss of specific enzymes.

By exposing the fungus to X-rays, Beadle and Tatum were able to induce mutations and create strains of the fungus that were unable to produce certain enzymes. They then performed biochemical tests to determine which metabolic pathways were affected in these mutant strains.

Through their experiments, Beadle and Tatum found that each mutation in a specific gene led to the loss of a single enzyme. This suggested a direct correlation between genes and enzymes, and they proposed the one gene one enzyme hypothesis based on their findings.

This hypothesis not only provided a link between genetic information and protein synthesis but also laid the foundation for the field of molecular biology. It opened up new avenues of research and led to further discoveries on the relationship between genetics and biochemistry.

The significance of the one gene one enzyme hypothesis in biochemistry

The one gene one enzyme hypothesis, proposed by George Beadle and Edward Tatum in 1941, revolutionized the field of biochemistry. It states that each gene is responsible for producing a specific enzyme, which in turn carries out a specific biochemical reaction in the body.

This hypothesis was a major breakthrough as it provided a direct link between genes and proteins. Prior to this hypothesis, the exact role of genes in protein synthesis was not fully understood. The one gene one enzyme hypothesis helped unravel the intricate relationship between genes, enzymes, and biochemical pathways.

The significance of this hypothesis lies in its implications for understanding genetic diseases. By studying the effects of mutations in genes and the subsequent loss or alteration of specific enzymes, scientists can gain insights into the underlying causes of various genetic disorders. This knowledge can then be used to develop targeted therapies and interventions.

Furthermore, the one gene one enzyme hypothesis paved the way for numerous advancements in biotechnology. It allowed researchers to manipulate genes and enzymes to produce desired proteins, leading to the development of recombinant DNA technology and the production of important drugs, hormones, and enzymes on a large scale.

In summary, the one gene one enzyme hypothesis is a fundamental concept in biochemistry that has had a profound impact on our understanding of genes, enzymes, and their roles in biological processes. Its significance extends to fields such as genetics, medicine, and biotechnology, making it a cornerstone of modern molecular biology.

The relationship between genes and enzymes

The one gene one enzyme hypothesis is a concept that suggests that each gene is responsible for the production of a specific enzyme. Enzymes are proteins that act as catalysts in various biochemical reactions within the cells.

This hypothesis was proposed by George Beadle and Edward Tatum in the 1940s, after conducting experiments on the bread mold Neurospora crassa. They found that mutations in specific genes resulted in the loss of certain enzymes, leading to defects in metabolic pathways.

According to this hypothesis, genes contain the instructions for building enzymes. Each gene is responsible for producing a single enzyme that performs a specific function. This establishes a direct relationship between genes and enzymes, as a change in the gene sequence can lead to alterations in the enzyme’s structure or activity.

The one gene one enzyme hypothesis laid the foundation for understanding the relationship between genes and proteins. It provided evidence that genes encode the information required for the production of specific proteins, in this case, enzymes. However, it has been further refined to the one gene one polypeptide hypothesis, as not all proteins are enzymes and some enzymes are composed of multiple polypeptide chains.

Nonetheless, the one gene one enzyme hypothesis remains an important concept in molecular biology and has contributed significantly to our understanding of how genes regulate protein synthesis and function.

The implications of the one gene one enzyme hypothesis in genetic diseases

The one gene one enzyme hypothesis proposes that each gene is responsible for encoding a specific enzyme. This hypothesis, first proposed by George Beadle and Edward Tatum in the early 1940s, revolutionized our understanding of genetics and had significant implications for the field of genetic diseases.

Genetic diseases are caused by mutations in genes that result in the production of faulty enzymes or the complete absence of enzymes. The one gene one enzyme hypothesis suggests that understanding the specific gene responsible for encoding the defective enzyme in a genetic disease can provide insights into the underlying molecular mechanisms and potential treatment options.

By identifying the gene associated with a particular genetic disease, researchers can further investigate the function of the enzyme produced by that gene. This knowledge is crucial for understanding the biochemical pathways and processes that are disrupted in the disease. It also allows for the development of targeted therapies that aim to restore or replace the dysfunctional enzyme.

Additionally, the one gene one enzyme hypothesis emphasizes the importance of studying individual genes and their associated enzymes in order to fully comprehend the complexity of genetic diseases. This approach enables scientists to determine the precise genetic and molecular factors contributing to a particular disorder and provides a foundation for personalized medicine.

Furthermore, the one gene one enzyme hypothesis has paved the way for advancements in genetic testing and disease diagnosis. By analyzing the genes and their corresponding enzymes, medical professionals can identify specific genetic mutations or abnormalities that may be indicative of certain genetic disorders. This information allows for early detection, accurate diagnosis, and appropriate treatment strategies.

In conclusion, the one gene one enzyme hypothesis has had profound implications in the study of genetic diseases. It has provided a framework for understanding the relationship between genes and enzymes, as well as a basis for developing targeted therapies and advancing genetic testing and diagnosis. This hypothesis continues to shape our understanding of the complex nature of genetic diseases and holds promise for future discoveries and advancements in the field of genetics.

The applications of the one gene one enzyme hypothesis in biotechnology

The one gene one enzyme hypothesis is a fundamental concept in biology that states each gene codes for a single enzyme. This hypothesis has played a crucial role in various applications within the field of biotechnology.

One of the key applications of the one gene one enzyme hypothesis is in the field of genetic engineering. By understanding that each gene encodes for a specific enzyme, scientists can manipulate and modify gene sequences to produce desired enzymes with specific functions. This allows for the development of new enzymes that can be used in various biotechnological processes, such as the production of biofuels, pharmaceuticals, and agricultural products.

Another application is in the development of gene therapy. Gene therapy aims to treat and cure genetic diseases by replacing or modifying faulty genes. The one gene one enzyme hypothesis provides a foundation for this approach, as scientists can identify the specific gene responsible for the defective enzyme and work on correcting it. By introducing functional genes, they can restore the enzyme’s activity and potentially alleviate the symptoms of the disease.

The one gene one enzyme hypothesis has also been instrumental in the field of bioprocessing. Bioprocessing involves the use of biological organisms or their components to produce valuable products. By understanding the relationship between genes and enzymes, bioprocess engineers can optimize the production of enzymes using various organisms, such as bacteria or yeast. This allows for the efficient and cost-effective production of enzymes for industrial applications, such as in the food and beverage industry, detergent production, and waste treatment.

In conclusion, the one gene one enzyme hypothesis has revolutionized the field of biotechnology. Its applications in genetic engineering, gene therapy, and bioprocessing have paved the way for advancements in various industries and have the potential to greatly benefit society.

The validity of the one gene one enzyme hypothesis

The one gene one enzyme hypothesis is a fundamental concept in molecular biology. It suggests that each gene is responsible for the production of a specific enzyme. This hypothesis was proposed by George Beadle and Edward Tatum in the early 1940s, based on their experiments with the bread mold Neurospora crassa .

Although the one gene one enzyme hypothesis was groundbreaking at the time, it is now known to be an oversimplification. It is important to understand that genes do not always code for enzymes. Genes can also code for various other functional molecules such as structural proteins, transport proteins, and regulatory molecules.

Furthermore, recent advancements in molecular biology have revealed that a single gene can code for multiple proteins through alternative splicing. This means that different combinations of exons can be included or excluded during the transcription process, leading to the production of different protein isoforms from a single gene.

Despite these complexities, the concept of the one gene one enzyme hypothesis still provides a valuable framework for understanding the relationship between genes and enzymes. It demonstrates the concept of gene expression and the notion that specific genes are responsible for specific biological functions.

Limitations of the hypothesis

However, it is important to recognize the limitations of the one gene one enzyme hypothesis. One of the major limitations is the idea that each gene codes for a single protein. This is not always the case, as some genes can code for multiple proteins through alternative splicing.

Another limitation is that the one gene one enzyme hypothesis does not account for post-translational modifications. After translation, proteins can undergo various modifications such as phosphorylation, glycosylation, and acetylation, which can alter their function or activity. These modifications are not directly determined by the gene sequence but are influenced by cellular factors and signaling pathways.

In conclusion, while the one gene one enzyme hypothesis may be a simplified model, it has provided a foundational understanding of gene-function relationships. It has paved the way for further research and discoveries in molecular biology. The validity of the hypothesis lies in its ability to explain the general principles of gene expression and the idea that specific genes are responsible for specific biological functions. However, it is essential to acknowledge the complexities and limitations of this hypothesis in light of new knowledge and advancements in molecular biology.

The future of the one gene one enzyme hypothesis

The one gene one enzyme hypothesis has been a cornerstone in the field of genetics, shaping our understanding of how genes are related to the synthesis of enzymes. This hypothesis, proposed by George Beadle and Edward Tatum in 1941, proposed that each gene is responsible for the production of a specific enzyme. This revolutionary idea paved the way for further research into the relationship between genes and enzymes, and it laid the foundation for our current understanding of genetic diseases and the role of enzymes in human health.

However, as our knowledge of genetics and molecular biology has advanced, the one gene one enzyme hypothesis has evolved. We now know that not all genes code for enzymes and that some genes can code for multiple enzymes. Additionally, we have discovered that genes can also code for other types of proteins, not just enzymes.

Expanding our understanding

As we continue to unravel the complexities of the human genome, new techniques and technologies are emerging that allow us to explore gene function in unprecedented detail. With the advent of techniques like CRISPR-Cas9, it is now possible to edit specific genes and study their effects on various cellular processes.

These advancements in gene editing and gene expression analysis have opened up new avenues of research to test and refine the one gene one enzyme hypothesis. By systematically altering specific genes and studying the resulting changes in enzyme production, we can gain a deeper understanding of the relationship between genes and enzymes.

Revisiting the hypothesis

While the one gene one enzyme hypothesis may no longer stand as an absolute truth, it continues to serve as a valuable framework for understanding the relationship between genes and enzymes. As we uncover more complexities and nuances in gene expression and protein synthesis, it will be essential to revisit and refine this hypothesis to incorporate new findings.

In the future, we can expect that our understanding of gene function and protein synthesis will continue to evolve. New research and discoveries will undoubtedly shed further light on the intricacies of gene and enzyme relationships, opening up new avenues for targeted therapies and treatments for genetic diseases.

• Overall, the future of the one gene one enzyme hypothesis is bright, as advancements in gene editing and molecular techniques enable us to further explore the relationship between genes and enzymes.
• We can expect that our understanding will become more nuanced and refined, leading to breakthroughs in personalized medicine and tailored treatments based on an individual’s unique genetic makeup.
• As we uncover more about how genes and enzymes interact, we will gain insights into the underlying molecular mechanisms that govern cellular processes and disease states.

In conclusion, while the one gene one enzyme hypothesis may have undergone modifications and expansions, it remains a fundamental concept in genetics. Studying gene-enzyme relationships will continue to be a crucial area of research, offering valuable insights into human health and disease.

The limitations of the one gene one enzyme hypothesis

The one gene one enzyme hypothesis, which states that each gene is responsible for producing one specific enzyme, has been a foundational concept in molecular biology. However, this hypothesis has some limitations that need to be addressed.

• Firstly, it is important to note that not all proteins are enzymes. While the one gene one enzyme hypothesis focuses on the relationship between genes and enzymes, it does not account for the production of other proteins that have important biological functions.
• Another limitation is that some enzymes are composed of multiple subunits, each of which can be encoded by a different gene. This means that one gene can be responsible for producing only part of an enzyme, while other genes are necessary for the complete enzymatic activity.
• In addition, some enzymes are known to have multiple isoforms, or slightly different versions, which can be encoded by different genes or arise from alternative splicing of the same gene. This adds another layer of complexity to the one gene one enzyme hypothesis.
• The one gene one enzyme hypothesis also does not account for post-translational modifications that can alter the activity or function of an enzyme. These modifications, such as phosphorylation or glycosylation, can be crucial for the regulation of enzyme activity and cannot be solely attributed to a single gene.
• Furthermore, the one gene one enzyme hypothesis does not consider the regulation of gene expression, which can result in the production of different enzymes under different conditions. Gene regulation mechanisms, such as transcription factors and epigenetic modifications, play a key role in determining which genes are expressed and therefore which enzymes are produced.

In conclusion, while the one gene one enzyme hypothesis has provided valuable insights into the relationship between genes and enzymes, it has its limitations. Understanding the complexity of protein production and function requires considering the various factors that can influence enzyme activity beyond the scope of a single gene.

The role of the one gene one enzyme hypothesis in protein synthesis

The one gene one enzyme hypothesis is a concept in genetics that postulates that each gene in an organism is responsible for the production of a single enzyme. This hypothesis was first proposed by George Beadle and Edward Tatum in the mid-20th century, based on their studies with the bread mold Neurospora crassa.

The one gene one enzyme hypothesis revolutionized our understanding of how genes control the synthesis of proteins. It provided a key insight into the relationship between genes and enzymes, and laid the foundation for the field of molecular genetics.

According to this hypothesis, genes are segments of DNA that contain the instructions for making specific proteins. The process of protein synthesis involves the transcription of DNA into messenger RNA (mRNA), which carries the genetic information from the nucleus to the ribosomes in the cytoplasm. The mRNA is then translated into a chain of amino acids, which folds into a specific three-dimensional structure to form a functional protein.

The one gene one enzyme hypothesis suggests that each gene codes for the production of a specific enzyme, which in turn catalyzes a specific chemical reaction in the cell. Enzymes are essential for various metabolic processes, such as digestion, cellular respiration, and DNA replication. By controlling the production of these enzymes, genes play a crucial role in maintaining the overall biochemical balance of the cell.

Further research has expanded upon the one gene one enzyme hypothesis, revealing that some genes code for proteins that are not enzymes. However, the fundamental idea that each gene has a specific function in protein synthesis remains a cornerstone of molecular genetics.

• Genes control the production of enzymes through the one gene one enzyme hypothesis.
• Protein synthesis involves the transcription of DNA into mRNA and translation into a chain of amino acids.
• Enzymes catalyze specific chemical reactions in the cell and play a vital role in metabolism.

The one gene one enzyme hypothesis and metabolic pathways

The one gene one enzyme hypothesis is a fundamental concept in genetics and biochemistry. It suggests that each gene is responsible for producing a single enzyme, which in turn is involved in a specific biochemical pathway.

This hypothesis was proposed by George Beadle and Edward Tatum in the 1940s based on their study of the bread mold Neurospora crassa. They discovered that mutations in specific genes resulted in the inability of the mold to produce certain enzymes, leading to defects in metabolic pathways.

What this means is that genes serve as instructions for the production of enzymes, which play crucial roles in the metabolism of cells. Enzymes are proteins that catalyze chemical reactions, making them essential for the breakdown and synthesis of molecules in various biochemical pathways.

Metabolic pathways are a series of interconnected chemical reactions that occur within cells. These pathways are responsible for converting raw materials into the energy and building blocks needed for cellular processes.

The one gene one enzyme hypothesis provides a framework for understanding how genes control the production of enzymes, which in turn regulate the metabolic pathways. By studying the relationship between genes, enzymes, and metabolic pathways, scientists can gain valuable insights into the functioning of cells and the underlying mechanisms of diseases.

The impact of the one gene one enzyme hypothesis on pharmaceutical research

The one gene one enzyme hypothesis is a fundamental concept in genetics that states that each gene is responsible for the synthesis of a specific enzyme. This hypothesis was first proposed by biochemists George Beadle and Edward Tatum in the 1940s.

Pharmaceutical research has greatly benefited from the understanding and application of the one gene one enzyme hypothesis. By identifying specific genes and their corresponding enzymes, scientists have been able to develop targeted therapies and medications for various diseases.

Understanding disease mechanisms

One of the key impacts of the one gene one enzyme hypothesis on pharmaceutical research is the ability to better understand the underlying mechanisms of diseases. By identifying the specific genes and enzymes involved in a particular disease, researchers can gain insights into the molecular pathways and processes that contribute to the development and progression of the disease.

With this knowledge, pharmaceutical companies can design drugs that specifically target these genes and enzymes, thereby interrupting or modulating the disease-related processes. This targeted approach has been successful in the treatment of various conditions, such as cancer, genetic disorders, and metabolic diseases.

Drug development and personalized medicine

The one gene one enzyme hypothesis has also revolutionized the field of drug development. By understanding the relationship between genes, enzymes, and diseases, researchers can identify new drug targets and develop more effective and specific therapies.

Furthermore, the concept of personalized medicine has become more attainable due to the one gene one enzyme hypothesis. With the identification of individual genes and their corresponding enzymes, scientists can tailor treatments to individuals based on their unique genetic profiles.

This personalized approach allows for more effective treatments with reduced side effects, as medications can be specifically designed to target the genetic variants present in each patient. This approach has already shown promising results in areas such as oncology and rare genetic diseases.

In conclusion, the one gene one enzyme hypothesis has had a profound impact on pharmaceutical research. It has provided scientists with a framework to understand the relationship between genes, enzymes, and diseases, leading to the development of targeted therapies and personalized medicine. This hypothesis continues to drive advancements in drug discovery and has the potential to revolutionize healthcare in the future.

The correlation between mutations and enzyme activity

The one gene one enzyme hypothesis proposes that each gene is responsible for producing a specific enzyme. This relationship between genes and enzymes implies that any changes or mutations in a gene can affect the activity and function of the corresponding enzyme.

When a mutation occurs in a gene, it can lead to a change in the sequence of amino acids in the protein encoded by that gene. Enzymes are proteins, and their function relies on the specific arrangement of amino acids in their structure. Therefore, even a single mutation can disrupt the enzyme’s structure and alter its catalytic activity.

The correlation between mutations and enzyme activity is crucial for understanding genetic diseases. In some cases, mutations can result in the loss or reduction of enzyme activity, leading to metabolic disorders. For example, in the genetic disorder phenylketonuria, a mutation in the gene that encodes the enzyme phenylalanine hydroxylase leads to a decreased activity of this enzyme, causing a buildup of phenylalanine in the body.

On the other hand, some mutations can enhance or change enzyme activity, leading to different physiological effects. These mutations can result in enzymatic variants with altered substrate specificity or increased catalytic efficiency. For instance, some mutations in the gene encoding the enzyme lactase allow individuals to digest lactose, the sugar found in milk, even in adulthood.

In conclusion, the one gene one enzyme hypothesis suggests that genes are responsible for producing specific enzymes. Mutations in genes can disrupt the structure and function of the corresponding enzymes, leading to changes in enzyme activity. Understanding the correlation between mutations and enzyme activity is essential for understanding and treating genetic diseases.

The role of the one gene one enzyme hypothesis in evolutionary biology

The one gene one enzyme hypothesis is a concept that proposes that each gene is responsible for encoding a single enzyme. This hypothesis, first formulated by George Beadle and Edward Tatum in the early 1940s, revolutionized our understanding of how genes function.

What is the significance of this hypothesis in the field of evolutionary biology? It provides a framework for understanding how mutations in genes can lead to changes in organisms over time. According to the one gene one enzyme hypothesis, changes in the DNA sequence of a gene can result in alterations to the structure or function of the corresponding enzyme. This, in turn, can lead to changes in an organism’s phenotype.

The central dogma of molecular biology

The one gene one enzyme hypothesis also fits into the central dogma of molecular biology, which describes the flow of genetic information within a biological system. According to this dogma, genetic information flows from DNA to RNA to protein. In the context of the one gene one enzyme hypothesis, genes code for enzymes, and enzymes play crucial roles in biochemical reactions within the cell.

Implications for evolutionary processes

The one gene one enzyme hypothesis has important implications for evolutionary processes. Mutations in genes can lead to the production of new enzymes or changes in the activity of existing enzymes. This variation in enzyme function can contribute to the survival and reproduction of individuals with advantageous traits, leading to evolutionary changes within populations over time.

In this table, each gene is associated with a specific enzyme and function. This supports the premise of the one gene one enzyme hypothesis, as it demonstrates the direct relationship between genes and enzymes.

In conclusion, the one gene one enzyme hypothesis has played a crucial role in shaping our understanding of genetics and evolutionary biology. It provides a framework for understanding how mutations in genes can lead to changes in enzymes and, ultimately, to changes in organisms over time. This hypothesis is an essential concept that forms the basis of many studies in molecular biology and genetics.

The involvement of the one gene one enzyme hypothesis in genetic engineering

The one gene one enzyme hypothesis is a fundamental concept in genetics that suggests that each gene is responsible for the production of a single enzyme. This hypothesis was proposed by George Beadle and Edward Tatum in the 1940s based on their experiments with bread mold. Their work demonstrated that each gene in the bread mold was responsible for the production of a specific enzyme, which played a crucial role in metabolic pathways.

This hypothesis has been instrumental in the field of genetic engineering. Genetic engineering involves the manipulation and modification of an organism’s genetic material to achieve desired traits or outcomes. By understanding the relationship between genes and enzymes, scientists are able to engineer organisms with specific traits by modifying their genes.

Example: Insulin production

An example of how the one gene one enzyme hypothesis is involved in genetic engineering is the production of insulin. Insulin is a hormone that regulates blood sugar levels, and its deficiency or malfunction can lead to diabetes. In the past, insulin for medical use was obtained from animal sources, such as pigs or cows, which posed risks of allergic reactions in some patients.

With the advancements in genetic engineering, scientists have been able to produce human insulin by modifying the gene responsible for insulin production. The human insulin gene is inserted into bacteria or other organisms, which act as production factories and synthesize human insulin. This approach ensures a more reliable and safer supply of insulin for diabetic patients.

The one gene one enzyme hypothesis has played a significant role in genetic engineering by providing a framework for understanding the relationship between genes and enzymes. By applying this hypothesis, scientists have been able to manipulate and modify genes to engineer organisms with desired traits, leading to advancements in various fields such as medicine, agriculture, and biotechnology.

The role of the one gene one enzyme hypothesis in the study of hereditary diseases

The one gene one enzyme hypothesis is a concept in molecular biology that suggests the relationship between genes and enzymes. It proposes that each gene is responsible for producing a specific enzyme, which in turn carries out a specific biochemical function. This hypothesis, formulated by George Beadle and Edward Tatum in the 1940s, has significantly influenced the study of hereditary diseases.

Hereditary diseases are genetic disorders that are passed down from parents to their offspring. They are caused by variations or mutations in specific genes, which can lead to the production of abnormal enzymes or the absence of essential enzymes. The one gene one enzyme hypothesis provides a framework for understanding the molecular basis of these diseases.

Molecular basis of hereditary diseases

Under the one gene one enzyme hypothesis, mutations in genes can result in the production of defective enzymes or the lack of necessary enzymes. This can disrupt normal physiological processes and lead to the development of various hereditary diseases.

For example, a mutation in a gene involved in the production of an enzyme responsible for breaking down a specific molecule could result in the accumulation of that molecule in the body. This accumulation can have detrimental effects on cellular functions and ultimately lead to a genetic disorder.

Impact on diagnosis and treatment

The one gene one enzyme hypothesis has played a vital role in the diagnosis and treatment of hereditary diseases. Understanding the specific gene-enzyme relationship allows researchers and clinicians to identify the underlying genetic cause of a disease and develop targeted therapies.

Genetic testing, which involves analyzing an individual’s DNA to identify mutations in specific genes, has become a fundamental tool in the diagnosis of hereditary diseases. By detecting these genetic variations, healthcare professionals can facilitate early intervention and tailor treatment plans to the individual’s specific needs.

Furthermore, the knowledge gained from studying the one gene one enzyme hypothesis has paved the way for the development of gene therapy and precision medicine. Gene therapy aims to correct or replace abnormal genes, while precision medicine utilizes an individual’s genetic information to customize treatment approaches. These advancements hold great promise for improving the management and outcomes of hereditary diseases.

In conclusion, the one gene one enzyme hypothesis has revolutionized our understanding of hereditary diseases. It has provided a crucial framework for unraveling the molecular basis of genetic disorders, enabling more accurate diagnosis and targeted treatment strategies.

The influence of the one gene one enzyme hypothesis on molecular biology

The one gene one enzyme hypothesis, proposed by George W. Beadle and Edward L. Tatum in the 1940s, revolutionized the field of molecular biology. This groundbreaking hypothesis suggested that each gene is responsible for coding a specific enzyme. By identifying the link between genes and enzymes, Beadle and Tatum laid the foundation for understanding the function and regulation of genes, as well as the biochemical pathways they govern.

Before the one gene one enzyme hypothesis, the relationship between genes and proteins was not well understood. It was known that genes controlled the production of proteins, but the exact mechanism was unknown. The one gene one enzyme hypothesis provided a crucial link between genes and proteins, establishing that each gene codes for a specific enzyme.

The significance of enzymes

Enzymes are biological catalysts that speed up chemical reactions in living organisms. They play a vital role in various metabolic pathways, including digestion, energy production, and DNA replication. Enzymes are essential for maintaining the overall function and homeostasis of an organism.

With the one gene one enzyme hypothesis, researchers gained a deeper understanding of how genes control enzyme production. This knowledge paved the way for studying the genetic basis of enzyme deficiencies and disorders. It also allowed scientists to investigate the regulation of gene expression and the impact of genetic mutations on enzyme function.

Advancements in molecular biology

The one gene one enzyme hypothesis marked a major milestone in the field of molecular biology. It provided a solid framework for the study of genetics, enzyme function, and metabolic pathways. This hypothesis led to further discoveries, such as the identification of specific genes responsible for diseases and the development of genetic engineering techniques.

Today, molecular biologists continue to build upon the one gene one enzyme hypothesis, exploring the intricate relationship between genes, enzymes, and proteins. The concept of one gene one enzyme has expanded to include a broader understanding of gene-protein interactions, post-translational modifications, and regulatory mechanisms.

The effects of the one gene one enzyme hypothesis in enzyme regulation

The one gene one enzyme hypothesis, proposed by George Beadle and Edward Tatum in the early 1940s, revolutionized our understanding of how genes control the production of enzymes. According to this hypothesis, each gene is responsible for the production of a single enzyme.

This hypothesis has had a significant impact on our understanding of enzyme regulation. Enzymes are essential proteins that catalyze chemical reactions in living organisms. The regulation of enzyme activity is crucial for maintaining the proper functioning of biological processes.

Understanding the relationship between genes and enzymes allows us to study the mechanisms by which genes control enzyme activity. The one gene one enzyme hypothesis provides a framework for investigating how changes in gene expression can lead to changes in enzyme production and activity.

By studying the effects of mutations in specific genes, scientists can gain insights into the role of these genes in enzyme regulation. For example, if a mutation in a gene leads to the production of a non-functional enzyme, it suggests that the gene is responsible for encoding that particular enzyme.

Furthermore, this hypothesis has also paved the way for the development of techniques such as gene knockout and gene overexpression, which allow researchers to manipulate gene expression and study its effects on enzyme production and activity.

The one gene one enzyme hypothesis has expanded our understanding of the relationship between genes and enzymes. It has provided a foundation for studying enzyme regulation and has paved the way for further research into the complex mechanisms that control enzyme activity.

The relationship between the one gene one enzyme hypothesis and metabolic disorders

The one gene one enzyme hypothesis proposes that each gene is responsible for producing a specific enzyme. This hypothesis, first proposed by George Beadle and Edward Tatum in the 1940s, revolutionized our understanding of genetics and laid the foundation for further exploration of gene function.

Metabolic disorders, also known as inborn errors of metabolism, are genetic disorders that result from a deficiency or malfunction of specific enzymes involved in various metabolic pathways. These disorders can affect the metabolism of different substances, such as carbohydrates, proteins, or fats.

The one gene one enzyme hypothesis is closely linked to metabolic disorders, as it suggests that mutations or variations in specific genes can lead to the production of non-functional or altered enzymes. This can disrupt the normal metabolic processes and lead to the development of metabolic disorders.

For example, phenylketonuria (PKU) is a metabolic disorder caused by a mutation in the gene responsible for producing the enzyme phenylalanine hydroxylase. This enzyme is essential for the breakdown of the amino acid phenylalanine. In individuals with PKU, the absence or reduced activity of phenylalanine hydroxylase leads to the accumulation of phenylalanine in the body, which can cause intellectual disability and other symptoms if left untreated.

Similarly, other metabolic disorders such as Gaucher disease, Tay-Sachs disease, and cystic fibrosis are also caused by mutations in specific genes encoding enzymes involved in various metabolic pathways.

Understanding the genetic basis of metabolic disorders

Studying the relationship between the one gene one enzyme hypothesis and metabolic disorders has provided valuable insights into the genetic basis of these disorders. By identifying the specific genes and enzymes involved, researchers can better understand the underlying mechanisms and develop targeted treatments.

Advances in genetic testing and sequencing technologies have also made it possible to identify gene mutations associated with metabolic disorders, allowing for earlier diagnosis and intervention. This knowledge has greatly improved patient care and outcomes in the field of metabolic medicine.

Future implications and research directions

Continued research in this field is crucial for advancing our understanding of metabolic disorders and developing effective treatment strategies. Further exploration of the genes and enzymes involved in metabolic pathways can lead to the development of targeted therapies, gene therapies, or even potential gene-editing approaches.

Furthermore, the one gene one enzyme hypothesis continues to be an important concept in the study of genetics and biochemistry. While the hypothesis has evolved to become more nuanced over time, it remains a fundamental principle that has shaped our understanding of the relationship between genes, enzymes, and metabolic disorders.

• To summarize:
• The one gene one enzyme hypothesis proposes that each gene is responsible for producing a specific enzyme.
• Metabolic disorders result from a deficiency or malfunction of specific enzymes involved in metabolic pathways.
• The one gene one enzyme hypothesis is closely linked to metabolic disorders, as gene mutations can lead to non-functional or altered enzymes.
• Understanding the genetic basis of metabolic disorders has improved diagnosis, interventions, and patient care.
• Continued research in this field can lead to the development of targeted therapies and potential gene-editing approaches.

The one gene one enzyme hypothesis and the Human Genome Project

The one gene one enzyme hypothesis is the idea that each gene is responsible for producing a single enzyme, which carries out a specific function in the body. This hypothesis was first proposed by George Beadle and Edward Tatum in the 1940s, and it revolutionized our understanding of genetics.

The Human Genome Project, which began in the 1990s, aimed to map and sequence the entire human genome. This ambitious project was a major scientific undertaking that involved researchers from around the world. By identifying and sequencing all the genes in the human genome, scientists hoped to gain a better understanding of human genetics and the role that genes play in human health and disease.

The one gene one enzyme hypothesis was a foundational principle of the Human Genome Project. It provided the framework for understanding how genes and enzymes are related and how they function together in the body. By mapping and sequencing the human genome, scientists were able to identify thousands of genes and their corresponding enzymes, leading to important discoveries about genetic diseases and potential therapies.

The one gene one enzyme hypothesis and the Human Genome Project have greatly contributed to our knowledge of genetics and have paved the way for advancements in personalized medicine and gene therapy. This research continues to have a profound impact on our understanding of human biology and the treatment of genetic diseases.

The role of the one gene one enzyme hypothesis in enzyme classification

The one gene one enzyme hypothesis, proposed by George Beadle and Edward Tatum in 1941, revolutionized the field of enzyme classification. Enzymes are proteins that play a crucial role in catalyzing biochemical reactions in living organisms. Before the one gene one enzyme hypothesis, enzymes were poorly understood and their functions were not well defined.

According to this hypothesis, each gene is responsible for producing a specific enzyme. This means that a single gene is responsible for coding the amino acid sequence of one particular enzyme. The genes themselves are located on chromosomes and carry the information necessary for the production of enzymes.

This hypothesis provided a framework for understanding the relationship between genes and enzymes. By studying the effects of mutations in specific genes, scientists were able to identify the corresponding enzyme that was affected. This led to the development of enzyme classification based on their corresponding genes.

Enzyme classification became an essential tool in biochemistry and genetics research. By identifying the genes responsible for the production of specific enzymes, scientists could further investigate the functions and mechanisms of these enzymes. This knowledge has profound implications in various fields, including medicine, agriculture, and biotechnology.

The one gene one enzyme hypothesis laid the foundation for our understanding of the relationship between genes and enzymes. It allowed scientists to connect specific genes with the production of distinct enzymes, enabling further research in enzyme classification and the elucidation of their functions. This hypothesis remains a fundamental principle in molecular biology and continues to shape our understanding of the complex interactions between genes and proteins.

The contribution of the one gene one enzyme hypothesis to our understanding of genetics

The one gene one enzyme hypothesis, first proposed by American geneticist George Beadle in 1941, revolutionized our understanding of genetics. This hypothesis states that each gene is responsible for producing a specific enzyme, which in turn is responsible for a specific chemical reaction within the cell.

This groundbreaking idea provided a conceptual framework for understanding how genes control the biochemical processes of life. Prior to this hypothesis, the relationship between genes and enzymes was not well understood.

The one gene one enzyme hypothesis proposes that each gene is responsible for producing a specific enzyme. Enzymes are proteins that catalyze chemical reactions in the cell. This hypothesis suggests that genes control the production of enzymes, and therefore control the biochemical reactions that occur within living organisms.

According to this hypothesis, if a gene is mutated or absent, the corresponding enzyme will not be produced or will be produced in an altered form. This can lead to biochemical imbalances and potentially result in genetic disorders.

The significance of the one gene one enzyme hypothesis

The one gene one enzyme hypothesis laid the foundation for our understanding of the relationship between genes and their role in cellular processes. It provided valuable insights into the mechanisms of genetic control and helped scientists unravel the complexities of gene function.

This hypothesis also paved the way for the development of new techniques and tools in molecular biology. The understanding that genes are responsible for producing specific enzymes led to the development of methods for studying gene expression and regulation.

Furthermore, the one gene one enzyme hypothesis served as a basis for subsequent research in genetics and provided a framework for understanding the role of genes in human health and disease. It has greatly contributed to our knowledge of genetic disorders and has steered the development of targeted therapies.

Overall, the one gene one enzyme hypothesis has had a profound impact on our understanding of genetics. It has provided valuable insights into the relationship between genes and their functions, and has laid the groundwork for further studies in the field of molecular biology.

The applications of the one gene one enzyme hypothesis in medicine

The one gene one enzyme hypothesis is a theory that proposes that each gene in an organism is responsible for producing a specific enzyme. This concept has had significant applications in the field of medicine, allowing scientists to better understand and treat various genetic disorders.

One of the key applications of the one gene one enzyme hypothesis is in the field of diagnostics. By identifying the specific genes and enzymes associated with certain diseases, doctors can use this information to diagnose patients more accurately and quickly. This is particularly important in cases where genetic disorders are present, as traditional diagnostic methods may not always be effective.

Furthermore, the one gene one enzyme hypothesis has opened up opportunities for the development of targeted therapies. By understanding the specific genetic mutations that lead to enzyme deficiencies, researchers can develop drugs and treatments that specifically target these enzymes. This approach has proven successful in the treatment of certain enzyme deficiencies, such as in cases of phenylketonuria and Gaucher disease.

Another area where the one gene one enzyme hypothesis has made an impact is in the field of genetic engineering. By manipulating specific genes and enzymes, scientists are able to produce desired proteins and enzymes for various medical purposes. This includes the production of therapeutic proteins, such as insulin, through recombinant DNA technology.

In conclusion, the one gene one enzyme hypothesis has revolutionized the field of medicine by providing a framework for understanding the relationship between genes and enzymes. Its applications in diagnostics, targeted therapies, and genetic engineering have greatly advanced our ability to diagnose and treat genetic disorders. Further research in this area holds the potential to uncover even more applications and therapeutic possibilities.

The one gene one enzyme hypothesis, proposed by George Beadle and Edward Tatum in 1941, states that each gene codes for the production of a specific enzyme. This hypothesis was later modified to the one gene one polypeptide hypothesis, as it was found that not all proteins are enzymes.

Who proposed the one gene one enzyme hypothesis?

The one gene one enzyme hypothesis was proposed by George Beadle and Edward Tatum in 1941. They conducted experiments on the bread mold Neurospora crassa, discovering that mutations in certain genes led to the loss of specific enzyme activities.

Why was the one gene one enzyme hypothesis modified?

The one gene one enzyme hypothesis was modified to the one gene one polypeptide hypothesis because it was discovered that not all proteins are enzymes. Some proteins have structural or regulatory functions, and not all genes code for enzymes. This modification acknowledged the diversity of proteins in living organisms.

What was the significance of the one gene one enzyme hypothesis?

The one gene one enzyme hypothesis was a landmark discovery in the field of genetics. It provided evidence for the concept that genes control the production of specific proteins, laying the foundation for the understanding of the relationship between genes and enzymes. This hypothesis also played a crucial role in the development of molecular genetics and the field of genetic engineering.

How was the one gene one enzyme hypothesis tested?

The one gene one enzyme hypothesis was tested by studying mutations in the bread mold Neurospora crassa. Beadle and Tatum exposed the molds to various mutagens and then observed the effects of these mutations on the ability of the molds to synthesize certain enzymes. By correlating specific mutations with the loss of enzyme activity, they provided evidence for the one gene one enzyme hypothesis.

The one gene one enzyme hypothesis is the idea that each gene encodes the information for producing a specific enzyme. This hypothesis was proposed by George Beadle and Edward Tatum in the 1940s based on their experiments with the bread mold Neurospora crassa. They showed that mutations in specific genes led to the inability of the mold to produce certain enzymes, linking one gene to one enzyme.

How was the one gene one enzyme hypothesis formulated?

The one gene one enzyme hypothesis was formulated by George Beadle and Edward Tatum in the mid-20th century. They conducted experiments with the bread mold Neurospora crassa and discovered that mutations in specific genes correlated with the inability of the mold to synthesize certain enzymes. From these findings, Beadle and Tatum deduced that each gene is responsible for the production of a specific enzyme, leading to the formulation of the one gene one enzyme hypothesis.

What is the significance of the one gene one enzyme hypothesis?

The one gene one enzyme hypothesis played a crucial role in the development of modern genetics. It provided evidence for the direct link between genes and their functional products, enzymes. This hypothesis paved the way for further research into the relationship between genes and proteins, and ultimately led to the understanding that genes code for proteins, which perform various functions in living organisms. It also contributed to the understanding of genetic diseases caused by enzyme deficiencies.

Are there any exceptions to the one gene one enzyme hypothesis?

Yes, there are exceptions to the one gene one enzyme hypothesis. While it was initially believed that each gene codes for a single enzyme, it is now known that many genes can code for multiple isoforms of an enzyme or different subunits of a protein complex. Additionally, there are genes that do not code for enzymes at all, but instead, regulate the expression of other genes. Therefore, the one gene one enzyme hypothesis does not apply universally and has been modified to accommodate more complex gene-protein relationships.

How did the one gene one enzyme hypothesis contribute to the field of biochemistry?

The one gene one enzyme hypothesis had a significant impact on the field of biochemistry. It provided a framework for understanding the relationship between genes and their functional products, enzymes. This understanding formed the basis for studying the structure and function of proteins, the development of recombinant DNA technology, and the field of molecular biology as a whole. The one gene one enzyme hypothesis also highlighted the importance of studying genetic disorders caused by enzyme deficiencies, leading to advancements in medical research and treatments.

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7.3 The “One Gene: One Enzyme” Hypothesis

Beadle and Tatum’s experiments are important not only for their conceptual advances in understanding genes, but also because they demonstrate the utility of screening for genetic mutants to investigate a biological process — this is called genetic analysis .

Beadle and Tatum’s results were useful to investigate biological processes, specifically the metabolic pathways that produce amino acids. For example, Srb and Horowitz (1944) tested the ability of the amino acids to rescue auxotrophic strains. They added one of each of the amino acids to minimal medium and recorded which of these restored growth to independent mutants.

Watch the video below, BIOL 183: Beadle & Tatum’s One-Gene-One-Enzyme hypothesis , by Susan Bush (2020) at Metropolitan State University on YouTube, which explains the one – gene – one – enzyme hypothesis.

A convenient example is arginine. If the progeny of a mutagenized spore could grow on minimal medium only when it was supplemented with arginine ( Arg ), then the auxotroph must bear a mutation in the Arg biosynthetic pathway and was called an “arginineless” strain (arg-).

Synthesis of even a relatively simple molecule, such as arginine, requires many steps — each with a different enzyme. Each enzyme works sequentially on a different intermediate in the pathway ( Figure 7.3.1 ). For arginine (Arg), two biochemical intermediates are ornithine (Orn) and citrulline (Cit). Thus, mutation of any one of the enzymes in this pathway could turn Neurospora into an Arg auxotroph (arg-). Srb and Horowitz extended their analysis of Arg auxotrophs by testing the intermediates of amino acid biosynthesis for the ability to restore growth of the mutants ( Figure 7.3.2 ).

They found that only Arg could rescue all the Arg auxotrophs, while either Arg or Cit could rescue some ( Table 7.3.1 ). Based on these results, they deduced the location of each mutation in the Arg biochemical pathway (i.e., which gene was responsible for the metabolism of which intermediate).

The video below, Gene Interactions P1, by Michelle Stieber (2014) on YouTube, discusses gene interactions and related biochemical pathways.

Media Attributions

• Figure 7.3.1 Original by Deyholos (2017), CC BY-NC 3.0 , Open Genetics Lectures
• Figure 7.3.2 Original by Deyholos (2017), CC BY-NC 3.0 , Open Genetics Lectures

Bush, S. (2020, April 16). BIOL 183: Beadle & Tatum’s one-gene-one-enzyme hypothesis (video file). YouTube. https://www.youtube.com/watch?v=4nXX2djQVvI

Deyholos, M. (2017). Figures: 4. A simplified version of the Arg biosynthetic pathway… and 5. Testing different Arg auxotrophs for their ability to grow…(digital image). In Locke, J., Harrington, M., Canham, L. and Min Ku Kang (Eds.), Open Genetics Lectures, Fall 2017 (Chapter 3, p. 3). Dataverse/ BCcampus. http://solr.bccampus.ca:8001/bcc/file/7a7b00f9-fb56-4c49-81a9-cfa3ad80e6d8/1/OpenGeneticsLectures_Fall2017.pdf

Srb, A. M. & Horowitz N. H. (1944). The ornithine cycle in Neurospora and its genetic control. Journal of Biological Chemistry, 154 , 129-139. https://doi.org/10.1016/S0021-9258(18)71951-0

Stieber, M. (2014, April 12). Gene interactions P1 (video file). YouTube. https://www.youtube.com/watch?v=Fv7UtsPfF-A

Long Descriptions

• Figure 7.3.1 The arginine biosynthetic pathway, showing the organic structures of citrulline and ornithine as intermediates in arginine metabolism. These chemical reactions depend on enzymes, which are the products of three different genes: A, B and C. [Back to Figure 7.3.1 ]
• Figure 7.3.2 Three mutants are tested for their ability to grow on minimal media, or minimal media supplemented with ornithine, citrulline, or arginine. Depending on the gene that is not functional in the particular mutant, they may or may not thrive in various media, therefore only arginine could rescue all the arginine auxotrophs, while either ornithine or citrulline could rescue others. [Back to Figure 7.3.2 ]

Introduction to Genetics Copyright © 2023 by Natasha Ramroop Singh, Thompson Rivers University is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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Microbe Notes

Lock and Key Model- Mode of Action of Enzymes

Enzymes are biological catalysts. These are commonly proteins but also include RNA (ribozymes) molecules that catalyze chemical reactions by lowering the activation energy of a reaction. These are known to speed up the rate of a reaction millions of times faster than the reaction without enzymes. Nearly all biological reactions require enzymes to transform substrate into products. The substrate is the reactant molecule upon which enzymes act during a chemical reaction, and products are the substances formed as a result of a chemical reaction. A single reactant molecule can decompose to give multiple products. Similarly, two reactants can enter into a reaction to yield products. These are reusable even after the completion of the reaction. Chemical properties such as charge and pH are vital in enzymatic reactions.

Binding between enzymes and reactant molecules takes place in such a way that chemical bond-breaking and bond-forming processes occur more readily. Meanwhile, no change in ∆the G value of a reaction takes place, thereby not altering the energy-releasing or energy-absorbing process of the reaction. However, it lowers the energy of the transition state, the topmost unstable state where the activated complex is formed from reactants that later give products.

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Enzyme’s Active site and Substrate Specificity

Enzymes are relatively larger than the substrates, whose only a small fraction is involved in catalysis by reducing chemical activation energy, also known as the catalytic site, and the other portion for binding with the substrate and orienting them also known as the binding site. The catalytic site and binding site altogether form the active site of an enzyme. Usually, there are two active sites in an enzyme.

•  The active site of enzymes is a cleft portion, composed of a small number of a unique combination of amino acid residues, usually three to four in number, which make up only ~10-20% of the volume of an enzyme. 
• The remaining amino acids are used to maintain tertiary structure by proper scaffold folding through non-covalent interactions.
• Non-covalent interaction between enzyme and substrate in correct orientation favors their reaction. These interactions include hydrogen bonds , hydrophobic bonds, ionic interactions, and Van der Waal’s interactions.
• However, transient covalent bonds between enzymes and substrates are also formed during the time of reaction.
• Side chains of amino acids play an important role in highly specific three-dimensional conformation at the level of the active site. These are large or small, hydrophilic or hydrophobic, acidic or basic.
• The specific shape, size, and chemical behavior of enzymes are determined by the nature of amino acids and their 3D space in the active site.

Specificity is a distinctive feature of enzymes where they have a unique ability to choose an exact substrate from a group of similar chemical molecules. Their specificity towards their substrate varies to a different extent. These are of different types, namely: Bond specificity, Group specificity. Substrate specificity, Stereospecificity, Geometrical specificity, and Co-factor specificity.

Substrate specificity is also k/a absolute specificity for the enzyme’s specificity towards one substrate and one reaction. For e.g., Lactase acts on the B-1-4 glycosidic linkage of lactose to yield galactose and glucose. The restrictive nature of enzymes towards the choice of substrate can be attributed to the enzymatic activity of two oxidoreductase enzymes. Alcohol dehydrogenase uses its substrate alcohol while lactic acid dehydrogenase act on lactic acid. Although these two enzymes function with the mechanism of oxidation and reduction reaction, their substrates can’t be used interchangeably. This is because the different structure of each substrate prevents their fitting into the active site of the alternative enzyme.

In most cases, cofactors, the non-protein molecules, are required to ensure an efficient enzyme-facilitated chemical reaction. These function to bind with enzymes via either ionic interaction or covalent interactions. Metal ions (such as minerals) and co-enzymes (vitamin derivatives) are cofactors.

Lock and Key Model

A German scientist, Emil Fischer postulated the lock and key model in 1894 to explain the enzyme’s mode of action. Fischer’s theory hypothesized that enzymes exhibit a high degree of specificity towards the substrate. This model assumes that the active site of the enzyme and the substrate fit perfectly into one another such that each possesses specific predetermined complementary geometric shapes and sizes. Thus, the shape of the enzyme and substrate do not influence each other. This specificity is analog to the lock and key model, where the lock is the enzyme, and the key is the substrate. In certain circumstances, if a second substrate similar in shape and size to the primary substrate is made to bind to the enzyme, this second substrate also fits in the active site too.

How does Lock and Key Model work?

• Binding of the substrate(s) to the enzyme at their active site takes place, thereby forming an enzyme-substrate complex.
• Enzymes catalyze the chemical reaction to take place, which can either be a synthesis reaction (favors bond formation) or a decomposition reaction (favors bond breakage).
• As a result, the formation of one or more products takes place, and the enzymes are released for their reuse in the next reaction.

Limitations of Lock and Key Model

• It doesn’t explain how the enzyme-substrate complex is stabilized in the transition state.
• This model supposes the enzyme is a rigid structure whose shape does not change upon binding with a suitable substrate. However, this is not supported by recent research, which states that there is a change in conformation of the active site of the enzyme upon binding of substrate.
• It does not describe the condition for binding multiple substrates to the enzyme.

Later, it was found that enzyme specificity toward one substrate is not always true. Although there are enzymes that specifically bind with only one substrate, there are also enzymes that exhibit broad specificity towards different but similarly structured substrates, such as lipase that can bind to different types of lipids. Similarly, proteases such as trypsin and chymotrypsin can degrade multiple types of proteins. Thus, the lock and key model is flawed, and the induced fit model was introduced to give a more refined view of the mode of enzymatic action.

• Blanco, A., & Blanco, G. (2017). Medical Biochemistry. Academic Press. https://www.khanacademy.org/science/ap-biology/cellular-energetics/enzyme-structure-and-catalysis/a/enzymes-and-the-active-site
• https://www.biologyonline.com/dictionary/substrate-specificity
• https://www.britannica.com/science/protein/The-mechanism-of-enzymatic-action
• https://www.biologyonline.com/dictionary/lock-and-key-model
• https://study.com/learn/lesson/lock-key-model-vs-induced-fit-model.html
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_(McMurry)/26%3A_Biomolecules-_Amino_Acids_Peptides_and_Proteins/26.11%3A_Enzymes_and_Coenzymes
• https://en.wikibooks.org/wiki/Structural_Biochemistry/Protein_function/Lock_and_Key
• https://ib.bioninja.com.au/higher-level/topic-8-metabolism-cell/untitled-6/models-of-action.html

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Introduction to enzymes and their applications

Saurabh Bhatia Published September 2018 • Copyright © IOP Publishing Ltd 2018 Pages 1-1 to 1-29

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Amity institute of Pharmacy, Amity university. Gurgaon, Haryana, India

Published September 2018

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Enzyme catalysis is an area of fundamental importance in different areas. This chapter offers a concise overview to the fundamental principles and mechanisms of action, catalysis inhibition and its pharmaceutical applications. Additionally, this section also covers basics information related with enzymes such as its structure, function and different properties.

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1.1. Introduction

The cell is the structural and functional unit of life—the basic building block of living systems. Cells have the capability to effectively utilize biocatalysts, known as enzymes, which have outstanding catalytic efficiency and both substrate and reaction specificity. Enzymes have amazing catalytic power and their high level of specificity for their substrate makes them suitable for biological reactions. They are crucial for cellular metabolism. Each and every chemical reaction that takes place in plants, micro-organisms and animals proceeds at a quantifiable rate as a direct result of enzymatic catalysis. Most of the history of biochemistry is directly or indirectly related to the history of enzyme research. Catalysis in biological systems was initially reported in the early 1800s based on research into the digestion of meat. In this report the catalytic activity of secretions from the stomach, the conversion of starch into sugar by saliva, and various plant extracts were reported.

In 1837, Berzelius documented the catalytic nature of fermentation. In the 1850s Louis Pasteur reported that fermentation was a process initiated by living organisms. During this study it was reported that the fermentation of sugar into alcohol by yeast was catalyzed by ferments. He also hypothesized that these ferments are close to the structure of yeast. These ferments were later called enzymes (in yeast). The key breakthrough in the history of enzymes came in 1897 when Edward Buchner isolated, from yeast cells, the soluble active form of the set of enzymes that catalyzes the fermentation of sugar to alcohol. Emul Fischer reported the first systematic studies on enzyme specificity in the early twentieth century [ 1 ]. Later, in 1926, James Sumner extracted urease in pure crystalline form from jack beans [ 2 ]. He also recognized the protein nature of urease . In 1930, John Northrop and his co-workers crystallized pepsin and trypsin and established them as proteins [ 3 ]. In subsequent years enzymology developed rapidly (table 1.1 ). The important developments during this period are: the elucidation of major metabolic pathways, such as the glycolysis and tricarboxylic acid cycle; the detection of numerous biochemical events of digestion, coagulation, muscular contraction and endocrine function, and their roles in the maintenance, control and integration of complex metabolic processes; the kinetic backgrounds to explain the observations of enzyme action and inhibition; and the development of protocols for examining the structures of functionally sensitive proteins. There has been exhaustive research on enzyme-catalyzed reactions and enzymes involved in cell metabolism. At present, 2000 different enzymes have been recognized, each of which catalyzes a different chemical reaction. Currently, more focus is being directed towards the application of enzymes. The high efficiency of enzymes makes them commercially valuable and their specificity of action is offering diverse advantages in clinical medicine.

Table 1.1.   Chronology of enzyme studies.

1.2. Properties of enzymes

Enzymes are the complex protein molecules, often called biocatalysts, which are produced by living cells. They are highly specific both in the reactions that they catalyze and in their choice of reactants, which are known as substrates. An enzyme typically catalyzes a single chemical reaction or a set of closely related reactions [ 4 ]. Side reactions resulting in the wasteful formation of by-products are rare in enzyme-catalyzed reactions, in comparison to uncatalyzed ones. Enzymes can also be defined as soluble, colloidal and organic catalysts that are produced by living cells, but are capable of acting independently of the cells [ 4 ]. Enzymes are currently being used in diverse areas in the food, feed, paper, leather, agriculture and textiles industries, resulting in major cost reductions. Simultaneously, rapid scientific progress is now encouraging the chemistry and pharmacological industries to embrace enzyme technology, a trend supported by concerns regarding energy, raw materials, health and the environment. One of the most common advantages of enzymes is their ability to function continuously even after their removal or separation from the cells. This means that even after the separation of cells from in vivo environments, they continue to work efficiently under in vitro conditions; we can conclude that these biocatalysts remain in an active state even after their isolation. Principally, enzymes are non-toxic, biodegradable and can be produced in ample amounts by micro-organisms for industrial applications. In this chapter, the isolation, production, purification, utilization and application of enzymes (in soluble and immobilized or insoluble form) are discussed in detail. Procedures such as recombinant DNA technology and protein engineering are frequently used to produce more efficient and beneficial enzymes. The industrial production and utilization of enzymes is an important part of industry. Interdisciplinary collaboration between areas such as chemistry, process engineering, microbiology and biochemistry is required to develop the best possible enzyme technology, and eventually to achieve increased production and maintain the enzyme's physico-chemical properties under in vitro environments.

For catalytic action, small quantities of an enzyme are sufficient, where this quantity of enzyme is much smaller in comparison to its substrates. The overall concentration of substrate transformed per mass of enzyme is often very large. Without exception, all enzymes are proteinaceous and exhibit all the properties of a protein. The treatment of enzymes by extreme temperature or extreme pH, or by treatment with other denaturing agents, results in the complete loss of catalytic activity. Structural configurations such as the primary, secondary, tertiary and quaternary structures of enzyme proteins are essential for their catalytic activity. The degree of catalytic activity chiefly depends on the integrity of the enzyme's structure as a protein. As per reports, enzymes have molecular weights ranging from about 12 000 to over 1 million Da. A number of enzymes consist only of polypeptides and contain no chemical groups other than amino acid residues, e.g. pancreatic ribonuclease. Numerous enzymes require a specific, heat stable, low molecular weight organic molecule, known as a co-enzyme. Moreover, a number of enzymes require both a co-enzyme and one or more metal ions for activity. A complete biochemically active compound is formed by the combination of a catalytically active enzyme (also called the protein part) with a co-enzyme or a metal ion—this is called a holoenzyme. The protein part of a holoenzyme is called an apoenzyme. In this arrangement a co-enzyme may bind covalently or noncovalently to the apoenzyme. In certain enzymes the co-enzyme or metal ion is only loosely and transiently bound to the protein. However, in others it is tightly and permanently bound, in which case it is known as a prosthetic group. A prosthetic group signifies a covalently bound co-enzyme. According to reports, co-enzymes and metal ions are stable under heating, while the protein part of an enzyme (the apoenzyme), is denatured by heat.

Prosthetic groups may be classified functionally into two major classes: co-enzymes and co-factors. Co-enzymes may be considered to be biosynthetically related to the vitamins, such as the co-enzyme nicotinamide adenine dinucleotide (NAD) which is vital for cellular energy metabolism and integrates the vitamin niacin into its chemical makeup. Moreover, a co-enzyme may be considered as a co-substrate, experiencing a chemical transformation throughout the enzyme reaction (NAD is reduced to NADH), the reversal of which requires a separate enzyme, perhaps from a different cellular site. Co-enzymes might thus travel intra-cellularly between apo-enzymes and, by transferring chemical groupings, integrate several metabolic processes. Table 1.2 shows a list of the more common co-enzymes and their functions. In contrast to co-enzymes, co-factors, such as pyridoxal phosphate or hem groups, remain with one enzyme molecule and in conjunction complete a cycle of a chemical change brought about by one enzyme turnover [ 5 ]. Other enzymes, such as carboxypeptidase, require metal ions as co-factors, the divalent cations Mg 2+ , Zn 2+ and Mn 2+ being the most common; these are often called enzyme activators [ 6 ]. Table 1.3 lists several enzymes and their respective co-factors.

Table 1.2.   Several co-enzymes employed in the transfer of specific atoms or functional groups.

Table 1.3.   Several enzymes and their co-factors.

1.3. Catalysis

The role of a catalyst is to increase the speed of a chemical reaction. When the rate of a chemical reaction is governed by a soluble catalyst, which may result in a further increase in the rate of chemical reaction, it is called homogeneous catalysis. In this case catalysis occurs in a solution. When the catalyst is in a separate phase from the reactants, or when catalysis occurs on a insoluble surface or an immobilized matrix, it is known as heterogeneous catalysis. Enzymes are also called biological catalysts. These biological catalysts generally have the properties of homogeneous catalysts, however, a number of enzymes present in membranes are insoluble, and thus are called heterogeneous catalysts. Enzyme specificity is the absolute specificity of protein catalysts to identify and bind to only one or a few molecules. In this process the enzyme carries a defined arrangement of atoms in their active site to bind with the substrate. This active site on the enzyme should have a shape that accurately matches the substrates. Thus specificity is achieved when an enzyme with an active site binds with the chemical reactants (the substrates) at their active sites via weak bond interactions. To undergo a chemical reaction, this active site carries certain residues that form a temporary bond with the chemical reactants, termed the binding site, whereas the catalytic site carries the residues that are responsible for catalysis. Specificity is achieved when a substrate binds to an enzyme that has a defined arrangement of atoms in the active site. An enzyme always catalyzes a single type of chemical reaction, which involves the formation and breakdown of covalent bonds. Since they are specific to one particular reaction, this feature of enzymes is called reaction specificity, also known as absolute reaction specificity, i.e. no by-products are formed.

1.4. The structure of enzymes

Enzymes always act as catalysts and small quantities compared to their substrate are required to considerably increase the rate of chemical reactions, wherein the enzymes themselves experience no overall change [ 7 , 8 ]. In contrast to all true catalysts, an enzyme does not alter the ultimate equilibrium position of a reaction, which is thermodynamically determined, thus merely the rate of completion of equilibrium of a feasible reaction is augmented. In addition to catalytic properties, enzymes exhibit the physico-chemical behavior of proteins: their solubility, electrophoretic properties, electrolytic behaviors and chemical reactivity [ 7 , 8 ]. The primary structural configuration and catalytic action of enzymes is determined by the linear chain of amino acid residues linked via peptide bonds, which constitute a protein molecule. Localized folding of the primary structure is called a secondary structure, whereas the complete folding of the molecule is known as a tertiary structure. In contrast to these structural configurations, a quaternary structure is the agglomeration of several folded chains. The structural features of enzymes are shown in figures 1.1 and 1.2 . In contrast to traditional chemical catalysts, e.g. hydrogen ions, heavy metals or metal oxides, which are most effective in organic solvents, at very high temperatures or at extreme pH values, enzymes operate most efficiently under very mild conditions. When using enzymes, there are certain issues that require attention, such as deviation from homogeneous aqueous solutions, physiological pH and temperature, which can rapidly destroy enzyme activity. However, under normal conditions the increase in reaction rate is rarely matched by their non-protein counterparts.

Figure 1.1.  Structural features of enzyme.

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Figure 1.2.  Principle components of an enzyme.

1.5. Structural features: primary and secondary structures

Three-dimensional analysis of the amino acid sequence of lysozyme of hen's egg white has demonstrated some features essential for primary structure [ 9 , 10 ]. These are:

• •   Molecules derived from a similar source have a similar order of amino acid residues and appear to be random with no obvious predictability.
• •   Even though numerous enzymes are intramolecularly crosslinked via disulfide bridges of cysteine, no branching occurs.

Current databases suggest that a small number of amino acids are extra and most are 'functional', i.e. the majority of them co-operatively control the higher orders of structural organization and therefore the catalytic activity. When comparing the primary structures of enzymes performing similar functions, wide structural homologies are detected in their sequence, mainly in the patterns of their nonpolar residues. For example, pancreatic juice contains five inactive precursors (zymogens), namely chymotrypsinogen A, B and C, trypsinogen and proelastase; all of these are activated to the respective proteases by proteolytic cleavage [ 11 ].

1.6. The mechanism of action of enzymes

The mechanism of action is based on a chemical reaction, in which the enzyme binds to the substrate and finally forms an enzyme–substrate complex. This reaction take place in a relatively small area of the enzyme called the active or catalytic site. In other words, the mechanism of enzyme action is based on the nature of the enzyme–substrate interaction, which accounts for the reaction specificity of the biological catalysts. The active or catalytic site of an enzyme is constituted by several amino acids, located at some distance from each other in the peptide chain. These amino acids are brought close together by the folding resulting from the secondary and tertiary structure of the enzymes. Side chains of amino acid residues at the catalytic site provide groups for binding with specific groups of the substrate. Co-factors assist the catalysis. The substrate forms bonds with amino acid residues in the substrate binding domain of the active site. The binding induces a conformational reaction in the active site. During the reaction, the enzyme forms a transition-state complex. As the products of the reaction disassociate, the enzyme returns to the original state. Two different models postulated for the mechanism of enzyme action are given below.

1.6.1. The Fisher template model (lock and key model)

This is a rigid model of the catalytic site, proposed by Emil Fischer in 1894 [ 12 ]. The model explains the interaction between a substrate and an enzyme in terms of a lock and key analogy. In this model, the catalytic site is presumed to be preshaped. The substrate fits as a key fits into a lock. The drawback of this model is the implied rigidity of the catalytic site. The model cannot explain changes in enzyme structure in the presence of allosteric modulators.

1.6.2. Induced fit model

In contrast to the above method, this model suggests a flexible mode for the catalytic site. To overcome the problems of the lock and key model owing to the rigid catalytic site, Koshland [ 13 – 15 ] suggested an induced fit model in 1963. The important feature of this procedure is the flexibility of the active site. In the induced fit model, the substrate induces a conformational change in the active site of the enzyme so that the substrate fits into the active site in the most convenient way so as to promote the chemical reaction. This method suggests competitive inhibition, allosteric modulation and inactivation of enzymes on denaturation.

The Michaelis–Menten theory of enzyme action [ 16 ] offers the basis for most current research on the mechanism of enzyme action. This concept of the enzyme–substrate complex scheme assumes the combination of the enzyme and substrate in phase one (occasionally known as the transition phase) of the enzyme activity and liberation of the enzyme and the products of the catalysis in phase two of the reaction.

1.6.3. Covalent catalysis

Covalent catalysis is evidenced in enzymes capable of forming covalent bonds between the substance and the catalytic group of the active site [ 17 ]. A number of enzymes react with their substrates to form very unstable, covalently joined enzyme–substrate complexes, which undergo further reaction to yield products much more readily than in an uncatalyzed reaction. Several of the enzymes that exhibit covalent catalytic behavior are listed in table 1.4 .

Table 1.4.   Various enzymes exhibiting covalent catalytic behavior.

1.7. Catalysis via chymotrypsin

Hummel and Kalnitzky suggested an enzyme mechanism through the depiction of the sequential transition states experienced by the enzyme–substrate complex during catalysis [ 18 ]. Chymotrypsin is a digestive enzyme, responsible for proteolysis (breakdown of proteins and polypeptides) in the duodenum. Chymotrypsin favorably breaks peptide amide bonds (the carboxyl side of the amide bond is a large hydrophobic amino acid). These amino acids contain an aromatic ring in their side chain that fits into a 'hydrophobic pocket' of the enzyme. It is stimulated in the presence of trypsin. Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity [ 19 , 20 ].

1.7.1. Intermediary stages of chymotrypsin

As discussed above, chymotrypsin is a protease enzyme that cuts on the C-terminal phenylalanine, tryptophan and tyrosine on peptide chains [ 21 ]. Additionally, it is more specific for aromatic amino acids because of its hydrophobic pocket. Comparable to other serine proteases, chymotrypsin also catalyzes the hydrolysis of certain esters [ 22 ]. The molecular events involved in catalysis are called intermediary enzymology. Chymotrypsin, a protease, favorably accelerates breakdown of peptide bonds in which the aromatic amino acid (Phy, Try, or Trp) or bulky nonpolar R group (Met) contribute a carboxyl group. The synthetic substrate p-nitrophenyl acetate allows colorimetric analysis of chymotrypsin activity, as hydrolysis to p-nitrophenol, which is alkali, changes into the chromophore anionic forms.

1.7.2. Kinetic behavior of α -chymotrypsin

The kinetics of chymotrypsin of p-nitrophenyl acetate can be considered in a 'stop-flow' apparatus. This procedure utilizes substrate quantities of enzymes and measures the events in the first few milliseconds [ 23 ]. The use of p-nitrophenyl acetate as a substrate offers the prospect of investigating solvent effects on both the acylation of the enzyme and the hydrolysis (deacylation) of the acyl enzyme [ 23 ]. The significant features of the slow-flow kinetics of chymotrypsin are:

• - a burst phase featuring rapid liberation of an anion.
• - a subsequent 'steady-state' phase, with slower release of extra anion.
• •   A 'charge relay network' acts as a proton shuttle during catalysis by chymotrypsin. The charge relay network of chymotrypsin encompasses three aminoacyl residues that are far apart in a primary structural sense, but close together in a tertiary structural sense. While most of the charged residues of chymotrypsin are present at the surface of the molecule, those of the charge relay network are hidden in the otherwise nonpolar inner side of the protein. These charges transmit residues which activate sequential proton shifts that shuttle protons in the opposite direction. An equivalent series of proton shifts is assumed to accompany the hydrolysis of the physiologic chymotrypsin substrate, e.g. a peptide.

1.7.3. Selective proteolysis in creation of the catalytic sites of enzymes

Various enzymes, hormones and other physiologically active proteins are produced as inactive precursors (zymogens) that are further transformed to the active form by selective enzymatic cleavage (limited proteolysis) of peptide bonds. The final step to activating enzymatic function is limited proteolysis, either in a single activation step or in a consecutive series (cascade). The specificity of each activation reaction is evaluated by the complementarity of the zymogen substrate and the active site of the attacking protease. The arrangement of successive activation reactions is controlled by the specificity of each enzyme, while the extent of amplification of the initial stimulus is evaluated by the effectiveness of each activating step. Zymogen activation produces a prompt and irreversible response to a physiological stimulus, and is capable of initiating new physiological functions. Classical examples are the processes of hormone production, fibrinolysis, complement activation, blood coagulation, supra-molecular assembly, metamorphosis, fertilization and digestion. The zymogens of the pancreatic serine proteases, in particular, have functioned as models for detailed studies of the nature of the molecular changes that are involved in the intense increase in enzymatic activity that results upon incomplete proteolysis of the zymogen.

Specific proteolysis is a common means of activating enzymes and other proteins in biological systems. A number of proteins are manufactured and released in the form of inactive precursor proteins called proproteins. Various enzymes attain full enzymatic activity as they suddenly fold into their characteristic three-dimensional forms. In contrast, other enzymes are produced as inactive precursors that are successively activated by breakdown of one or a few specific peptide bonds. The inactive precursor is known as a zymogen (or a pro-enzyme). In other words, when the proteins are enzymes, the proteins are called pro-ezymes or zymogens (table 1.5 ). An energy source (ATP) is not required for cleavage [ 11 ]. Thus, in comparison to reversible regulation by phosphorylation, even proteins sited outside cells can be triggered by this means. An additional noteworthy difference is that proteolytic activation, in comparison with allosteric control and reversible covalent modification, occurs just once in the life of an enzyme molecule. Transformation of a proprotein to the mature protein includes selective proteolysis. This transforms the proproteins by one or more consecutive proteolytic clips to a arrangement in which the individual activity of the mature protein (its enzymatic activity) is expressed, e.g. the hormone insulin (proinsulin), the digestive enzyme chymotrypsin (chymotrypsinogen), a number of factors for blood clotting and for the blood clot dissolution cascades, and the connective tissue protein collagen (procollagen). Chymotrypsinogen consists of 245 amino acid residues, and is practically devoid of enzymatic activity. As the reaction starts, it is converted into a fully active enzyme. This occurs when the peptide bond joining arginine 15 and isoleucine 16 is cleaved by trypsin. The subsequent active enzyme, known as π-chymotrypsin, then acts on other π -chymotrypsin molecules. Two dipeptides are eliminated to form α -chymotrypsin (the stable form of the enzyme) [ 11 ]. The three subsequent chains in α -chymotrypsin remain interconnected to each another by two interchain disulfide bonds. The outstanding feature of this process is that cleavage of a single specific peptide bond alters the protein from a catalytically inactive form into one that is fully active. The transformation of prochymotrypsin (Pro-CT), a 2,4,5-aminoacyl residue polypeptide, to the active enzyme α -chymotrypsin includes three proteolytic clips and the formation of an active intermediate called π -chymotrypsin ( π -CT) and consequently to the mature catalytically active enzyme α -chymotrypsin ( α -CT). Examples of gastric and pancreatic zymogens are listed in table 1.5 .

Table 1.5.   Gastric and pancreatic zymogens.

1.7.4. Kinetic models for enzymes

Generally, enzyme kinetics is defined as the study of the rate of reactions, i.e., how the substrate concentration impacts the velocity of the reaction. Enzyme kinetics involves optimization of bio-catalytic reactions to allow process design and scaling up processes to further increase the production and minimize the overall overhead costs of various procedures. Kinetic investigations in the branch of biochemistry concerned with enzymes can be categorized into three types:

• •   Transient-state kinetics : This is the stage of reaction before the steady or rapid-equilibrium state, and involves quick reactions between the enzymes and substrate. These sudden changes in the reaction mixture when the substrate and enzymes are mixed require advance equipment to monitor the reaction before it changes into the steady state. The mechanisms of the reaction are associated with the enzyme structural configuration. Basic steps are involved during an enzyme-catalyzed reaction, which allow the direct study of the intermediates and products formed during a single enzyme cycle, which may further help in direct analysis of individual reaction steps for short times. In this type of reaction a sufficient concentration of enzymes is used to witness the intermediate and product formation.
• •   Steady-state kinetics : This is the phase in which the rate of formation of intermediates and the rate of decomposition remain the same, and thus the concentrations of reactive intermediates remain the same. During this reaction substrate concentration is greater than enzyme concentration. The Michaelis–Menten enzyme kinetic (figure 1.3 ) can be considered as the most often studied reaction for several enzymes. For example, chymotrypsin (protease) with a high concentration of substrate achieves maximum velocity of the reaction (called the first order of reaction) but at a certain point the substrate occupies all binding sites of the enzyme, after which further addition of substrate does not increase the rate. This is called the zeroth order of reaction (the steady state). It is the phase in which the enzyme and substrate concentrations cannot be determined using the dissociation constant. Thus steady-state enzyme kinetics is based on the theory that a catalytic reaction remains constant if the reaction is not exposed to continuous changes.
• •   Rapid-equilibrium kinetics : This the phase in which both the enzyme and substrate concentrations can be determined using the dissociation constant. During this procedure total enzyme concentration remains constant during the reaction and the concentration is very small compared to the amount of substrate. In this reaction, before the rate-determining reaction, the reactions are in equilibrium with their components, thus this stage is called rapid-equilibrium kinetics.

Figure 1.3.  The Michaelis–Menten enzyme kinetic.

According to reports, factors that affect enzyme-catalyzed reactions also affect the velocity of a reaction. These factors are called modifiers of enzyme-catalyzed reactions. These modifiers can be divided into two classes: inorganic modifiers (enzyme activators) and organic modifiers (enzyme inhibitors). These factors can have different types of effects on the velocity of the reaction; nevertheless the most vital effect is that they offer many pathways to products, e.g. when one modifier is bound to an enzyme, it alters the rate of reaction and thus forms two rate constants. However, when two modifiers participate, there are five self-regulating equilibria, resulting in three paths for making products.

There are two mechanisms, single-substrate and multiple-substrate, that are helpful in studying the different stages of enzymatic reactions. Understanding these stages helps in understanding the properties of enzymes. Certain enzymes have single substrates (a single substrate binding site), e.g. triosephosphate isomerase, whereas certain enzymes have multiple substrates molecules (multiple binding sites), such as dihydrofolate reductase, and bind with multiple substrates. After the exploration of specific RNA sequences required for RNA replication, new biocatalysts in the form of ribozymes have emerged with the potential to catalyze specific biochemical reactions. There is a misconception about biological catalysts that all biological catalysts are made up of proteins, which is not true; some are RNA-based catalysts (ribozymes and ribosomes). Both are important for many cellular functions. A major difference between enzymes and ribozymes is that RNA-based catalysts are restricted to only a few reactions; however, their reaction mechanisms and kinetics can be studied and classified by similar procedures. Enzyme-based mutation, in particular site-directed mutagenesis, is an important approach to alter genes and investigate the functional and structural features of enzymes, e.g. mutation of the enzyme present in Coprinus cinereus peroxidase offers an understanding of its increased thermostability. Challenges involved in studying cascades of reactions catalyzed by a multi-enzyme, e.g. proteasome involved in the ubiquitin–proteasome pathway, can be overcome by establishing understanding of the complex structure and the respective biochemical reactions. This understanding allows exploration of active sites, intermediate compounds, final products and their interrelation with complex machinery, as well as biochemical reactions. It has been well understood that enzymes that accelerate complex reactions have numerous substrates and involve complex enzyme kinetic mechanisms. As discussed above, most of the biochemical reactions occurring in the body are multi-substrate reactions. In such reactions two substrates are involved and yield two products (figure 1.4 ). These types of reactions involve the transfer of a compound from one compoment to another, e.g. when glucose reacts with ATP in the presence of hexokinase it forms glucose 6-phophaste and ADP. Here, phosphate from ATP is transfered to glucose to form glucose 6 phosphate. The mechanism of catalysis involves two types of reactions: sequential and non-sequential reactions. Sequential reaction results in the formation of a ternary complex. This means that both of the substrates involved in the reaction bind with an enzyme to form the product (figure 1.4 ). Sequential reaction is further divided into two types: the random and compulsory order mechanisms. As the name suggests, in a 'random' mechanism, either substrate can bind first and any product can leave first. In contrast to the random order mechanism, in the compulsory order mechanism the order of binding of the substrate and order of release of the product is specific; this is also called the Theorell–Chance mechanism (figure 1.4 ). In a non-sequential reaction, also called the 'ping-pong' mechanism, formation of ternary complex does not take place. In these types of reactions, when the first substrate binds with enzyme its product is released, and then the second substrate binds and its product is released. Such a reaction is called a double placement reaction. Thus only a single substrate binds at a time; this may be due to the presence of a single binding site on the enzyme. Major differences between the sequential and non-sequential reactions are that the formation of a ternary complex takes place only in the sequential reaction, and that in the sequential reaction both substrates bind to the enzyme and release products, while in the non-sequential mechanism the substrates bind and release their products one after the other (figure 1.4 ).

Figure 1.4.  Multi-substrate reactions.

Another type of sequential mechanism is the systematic mechanism, which involves the addition of substrates and formation of products in a specific order.

1.7.5. Enzyme mediated acid–base (general) catalysis

Several protein enzymes use general acid–base catalysis as a way to increase reaction rates [ 26 ]. The amino acid histidine is optimized for this function because it has a pK(a) (where K(a) is the acid dissociation constant) near physiological pH [ 26 ].

When the substrate has been bound at the catalytic site, the charged functional groups of the side chains of neighboring aminoacyl residues may contribute in catalysis by behaving as acidic or basic catalysts. There are two extensive groups of acid–base catalysis by enzymes: general and specific (acid or base) catalysis. Specific acid or specific base catalysis are those reactions in which the reaction rates fluctuate under the influence of changes in H + or H 3 O + concentration, but are independent of the concentrations of the other acids or bases present in the solution. In contrast to specific catalysis, general acid or general base catalysis are the reactions whose rates are very reactive to all acids (proton donors) or bases (proton acceptors) present in the solution. To examine whether a given enzyme-catalyzed reaction is a general or specific acid or base catalysis, the rate of reaction is determined under two sets of circumstances:

• •   at different pH values at a constant buffer concentration, and
• •   at constant pH values but at different buffer concentrations. Against this background, if the degree of the reaction deviates as a function of pH at a constant buffer concentration, the reaction is specific base/acid catalyzed if the pH is above/below 7.0. If the reaction rate at a constant pH rises as the buffer concentration increases, the reaction is general base/acid catalysis, if the pH is above/below 7.0.

1.7.6. Metallozymes

Almost 25% of all enzymes include tightly bound metal ions or need them for activity. The major role of these metal ions is investigated using techniques such as x-ray crystallography, magnetic resonance imaging (MRI) and electron spin resonance (ESR). A metalloprotein is a protein that contains a metal ion co-factor. Metallozymes contain a certain amount of functional metal ion that is retained during the course of purification [ 27 ]. A metal-activated enzyme binds with metals less firmly, but needs to be activated by addition of metals. Four types of complexes are possible for the tertiary complexes of the catalytic site (Enz), a metal ion (M) and substrate (S) that exhibit 1:1:1 stoichiometry:

All of these complexes are possible for metal-activated enzymes. Metallozymes cannot form the EnzSM complex (substrate–bridge complexes), as the purified enzyme exists as Enz–M. Three generalization can be made:

• •   The majority of the kinases (ATP: phosphotransferases) form substrate–bridge complexes of the type enzyme–nucleotide–M.
• •   Phosphotransferases (phosphoenolpyruvate or pyruvate used as the substrate), enzymes catalyzing other reactions of phosphoenolpyruvate and carboxylases, form metal bridge complexes (Enz–M–S).
• •   A particular enzyme may form one type of bridge complex with one substrate and a different type with another.

The metal ions participate in each of the four mechanisms by which the enzymes are known to accelerate the rates of chemical reaction:

• •   Approximation of reactants.
• •   Covalent catalysis.
• •   General acid–base catalysis.
• •   Induction of strain in the enzyme or substrate.

Metal ions are electrophiles (attracted to electrons) and share an electron pair forming a sigma bond. They may also be considered as super acids as they exist in neutral solutions, frequently having a positive charge which is greater than their quantity. Mn 2+ , Ca 2+ and Mg 2+ are the metal ions that are most commonly used in enzymatic catalysis. Two metal ions, iron and manganese are used in the form of haemprotein. Metal ions have the potential to accept electrons via sigma or pi bonds to successively activate electrophiles or nucleophiles. By means of donating electrons, metals can activate nucleophiles or act as nucleophiles themselves. The co-ordination sphere of a metal may bring together the enzyme and substrate or form chelate-producing distortion in either the enzyme or substrate [ 28 ]. A metal ion may also mask a nucleophile and thus avoid an otherwise probable side reaction. Metals can also function as three-dimensional templates for the co-ordination of basic groups on the enzyme or substrate.

1.8. Enzyme inhibition

Enzyme inhibition decreases the activity of an enzyme without significantly disrupting its three-dimensional macromolecular structure. Inhibition is therefore distinct from denaturation and is the result of a specific action by a reagent directed or transmitted to the active site region. When low molecular weight compounds interfere with the activity of enzymes by partially reducing or completely inhibiting the enzyme activity either reversibly or irreversibly, it is known as enzyme inhibition. The compounds responsible for such inhibition are called enzyme inhibitors. To protect the enzyme catalytic site from any change, a ligand binds with a critical side chain in the enzyme. Chemical modification can be performed to test the inhibitor for any drug value. Studies of enzymes can yield much information about the following:

• •   A number of drugs useful in medicine, which seem to function because they can inhibit certain enzymes in malfunctioning cells.
• •   The convenience of elucidating metabolic pathways in cells.
• •   The mechanism of the catalytic activity.
• •   The nature of the functional group at the active site.
• •   The substrate specificity of the enzyme.

The pharmacological action of drugs is mainly based on enzyme inhibition, e.g. sulfonamides and other antibiotics. In the majority of cases the enzyme inhibited is known. The development of nerve gases, insecticides and herbicides is based on enzyme inhibition studies. There are two major types of enzyme inhibition: reversible and irreversible.

Reversible inhibitors efficiently bind to enzymes by forming weak non-covalent interactions, e.g. ionic bonds, hydrophobic interactions and hydrogen bonds. Reversible inhibitors do not form any strong chemical bonds or reactions with the enzyme, they are formed quickly and can easily be removed, in contrast to irreversible inhibitors. Reversible inhibition includes competitive inhibition, uncompetitive inhibition and noncompetitive inhibition. Irreversible inhibition includes group specific inhibition (reacts only to a certain chemical group), reactive substrate analogs (affinity label) and inhibitors that are structurally similar to the substrate and will bind to the active site, and mechanism-based inhibitors (enzymes transform the inhibitor into a reactive form within the active site).

1.9. Pharmaceutical applications

Currently, enzymes are often utilized for a broad range of applications such as: washing powders (e.g. proteases, lipases, amylases); textile manufacture (amylases and catalase to remove the starch); the leather industry (proteases to hydrolyze proteins); the paper industry; improvement of the environment; food production (enzyme-modified cheese/butter), processing (glucose oxidase for dough strengthening) and preservation; and medical applications. According to current reports, several enzymes are produced industrially and there are significant applications in the food industry (45% of use), detergent industry (35%), textiles industry (10%) and leather industry (3%). Details on the applications of individual enzymes are provided in table 1.6 .

Table 1.6.   Industrially produced enzymes from plant sources and their applications.

1.9.1. Diagnostic applications of enzymes

Enzymes have been used widely in diagnostic applications varying from immunoassays to biosensors. Enzyme immunoassay methods hold great promise for application under a wide variety of conditions. Under laboratory conditions they can be as sensitive as radio-immunoassays, but they can also be adapted as simple field screening procedures [ 29 , 30 ]. The examination of enzyme quantity in the extracellular body fluids (blood plasma and serum, urine, digestive juices, amniotic fluid and cerebrospinal fluid) are vital aids to the clinical diagnosis and management of disease. Most enzyme-catalyzed reactions occur within living cells, however, when an energy imbalance occurs in the cells because of exposure to infective agents, bacterial toxins, etc, enzymes 'leak' through the membranes into the circulatory system. This causes their fluid level to be raised above the normal cell level. Estimation of the type, extent and duration of these raised enzyme activities can then furnish information on the identity of the damaged cell and indicate the extent of injury. Enzyme assays can make an important contribution to the diagnosis of diseases, as a minute change in enzyme concentration can easily be measured. Determination of the changes in enzyme level thus offers a greater degree of organ and disease differentiation in comparison to other possible clinico-chemical parameters, e.g. albumin or gamma globulin. Currently, the diagnostic specificity of enzyme tests is such that they are limited primarily to confirming diagnosis, offering data to be weighed alonside other clinical reports, owing to lack of disease specific enzymes. Table 1.7 includes a number of diagnostically important enzymes which are most often examined in clinic laboratories [ 29 , 30 ].

Table 1.7.   Diagnostically significant enzymes.

1 B, brain; E, erythrocytes; H, heart muscle; Ht, hepatobiliary tract; I, intestinal mucosa; K, kidney; L, M, skeletal muscle; Pa, pancreas; P1, placenta; Pr, prostate gland; S, saliva.

1.9.1.1. Enzyme examinations in diseases of the liver and biliary

The diseases of the liver and gastrointestinal tract were among the first to which serum enzyme tests were applied. They have proved to be most effective owing to the large size of the organs and the wide range and abundance of enzymes [ 32 – 36 ]. The liver-based enzymes GOT, GPT and AP are examined to evaluate the site and nature of liver disease. LD, GGT, OCT and CHE are also examined. Several enzymes employed in the diagnosis of liver diseases along with their respective levels are listed in table 1.8 .

Table 1.8.   Liver diseases and enzymes used in diagnosis [ 32 – 36 ].

1.9.1.2. Enzyme applications in heart disease

According to previous reports, no single enzyme has yet been reported to cure myocardial damage. The discovery of serum glutamine oxalacetic acid transaminase determination (GOT) in 1954 was considered a significant step forward in the diagnosis of acute myocardial infarction. A mixture of results from assays of CPK (creatine phosphokinase), HBD ( α -hydroxybutyrate dehydrogenase) and GOT (glutamine oxalacetic acid transaminase)—each of which has been shown to be elevated in more than 90% of cases—is used for diagnostic purposes [ 37 – 39 ]. The level of CPK starts rising three to four hours after the initial onset of pain, followed in order by GOT and AST (HBD) which appear after approximately eight hours. The maximum levels are reached in the same sequence, CPK after 24 h, LD 1 after 36 h and AST after about two days. The rise in enzyme levels is fairly moderate, AST and CPK increase by four to ten times their respective normal levels and LD 1 is approximately five-fold higher than normal. An enzyme known as hyaluronidase (hyaluronate hydrolysis) has been reported to cure heart attack [ 38 ]. The activity of many enzymes including aldolase, malic dehydrogenase, isomerase and ICD may increase following myocardial infarction [ 38 ].

1.9.1.3. Diagnosis of muscle disease

Skeletal muscle disorders include diseases of the muscle fibers (myopathies) or of the muscle nerves (neurogenic disorders) [ 40 ]. In myopathies CPJ, LD, ALD, GOT and GPT levels are raised. In the case of neurogenic diseases and hereditary diseases, CPK is occasionally raised (2–3 fold) [ 40 ]. Damage to the muscle may be due to extensive muscular exercise, drugs, physical trauma, inflammatory diseases, microbial infection or metabolic dysfunction, or it may be genetically predisposed. In muscular disorders the level of CPK is elevated in serum with the highest frequency and is assayed in the diagnosis of these disorders. An additional useful assayed enzyme is acetyl cholinesterase (AChE), which is significant in regulating certain nerve impulses [ 41 ]. Various pesticides affect this enzyme, so farm labors are frequently tested to be sure that they have not received accidental exposure to significant agricultural toxins. There are number of enzymes that are characteristically used in the clinical laboratory to diagnose diseases. There are highly specific markers for enzymes active in the pancreas, red blood cells, liver, heart, brain, prostate gland and many of the endocrine glands [ 41 ]. From the time when these enzymes became comparatively easy to examine using automated techniques, they have been part of the standard blood tests that veterinarians and medical doctors are likely to need in the diagnosis and treatment/management of diseases.

1.9.2. Enzymes in therapeutics

Enzymes have two significant features that differentiate them from all other types of drugs. First, enzymes frequently bind and act on their targeted sites with high affinity and specificity. Second, enzymes are catalytic and convert numerous target molecules to the desired products. These two important features make enzymes specific and potent drugs that can achieve therapeutic biochemistry in the body that small molecules cannot. These features have resulted in the development of many enzyme-based drugs for a wide range of disorders [ 42 ]. Currently, numerous enzymes are used as therapeutic agents, owing to the following features:

• •   High specificity to their substrates.
• •   Proficient in producing the desired effect without provoking any side effects.
• •   Water soluble.
• •   Extremely effective in a biological environment.

Enzymes as therapeutic agents also have some serious disadvantages which restrict their application. Their bulky structure, due to their large molecular weight, excludes them from the intracellular domain. Owing to their high proteinaceous nature they are highly antigenic and are rapidly cleared from blood plasma. Extensive purification from pyrogens and toxins is essential for parenteral enzymes, which increases the cost. Table 1.9 lists some therapeutically important enzymes.

Table 1.9.   Therapeutically important enzymes.

1.9.2.1. Enzyme therapy of cancer

In traditional medicine, proteolytic enzymes derived from plant extracts have been used for a long time In addition to proteolytic enzymes from natural resources such as plants, 'modern' enzyme therapy includes pancreatic enzymes. Therapeutically, the use of proteolytic enzymes is partly based on scientific reports and is partly empirical [ 43 ]. Clinical evidence of the use of proteolytic enzymes in cancer studies has typically been obtained with an enzyme preparation comprising a combination of papain, trypsin and chymotrypsin. Earlier reports proved that enzyme therapy can reduce the adverse effects caused by radiotherapy and chemotherapy. There is also a report available that, in some types of tumors, survival may be sustained. The positive effects of systemic enzyme therapy appear to be based on its anti-inflammatory potential. Nevertheless, the exact mechanism of action of systemic enzyme therapy remains unsolved. The proportion of proteinases to antiproteinases, which is regularly used as a prognostic marker in cancer studies, is likely to be influenced by the oral administration of proteolytic enzymes, most likely via induction of the synthesis of antiproteinases. In addition, there are many alterations of cytokine composition during treatment with orally administered enzymes, which might be a sign of the efficacy of enzyme therapy [ 44 ].

Proteases and their inhibitors have long been studied in several tumor systems. However, out of numerous promising serine and metalloproteinase inhibitors, not a single one is included in oncology at present. The present exploration for active antiproteolytic agents is in contrast to the traditional approach, as evidenced by John Beard, who proposed the management of advanced cancer using fresh pancreatic extracts whose antitumor activity was based on their proteolytic potential.

The enzymatic treatment of tumors is based on the idea of denying the abnormal cells their essential metabolic precursors such as amino acids, nucleic acids and folates. A number of enzymes have been examined and evidenced as antitumor agents. l -serine dehydratase, l -arginase, carboxypeptidase G (folate depletion), l -asparaginase, l -methioninase, l -phenylalanine ammonia lyase, l -glutaminase, l -tyrosinase and xanthine oxidase have been studied for their anticancer activity. Enzyme preparations such as asparaginase (amidase), bromelain (protease) and chymotrypsin (protease) have also been studied as cancer treatments (table 1.9 ).

l -asparaginase is the most widely investigated enzyme. It has been reported in treatment against three neoplastic diseases, acute lymphoblastic leukemia, leukemic lymphosarcoma and myeloblastic leukemia. It deprives the cancerous cells of their nutritional asparagine supply. Asparagine is essential for protein synthesis, which takes place inside the cell, and decreased protein synthesis perhaps accounts for the immunosuppression and toxic effects of asparaginase-based treatment.

The prospects of enzyme-based treatment against cancer are very bright, but the difficulties of antigenicity and short circulation time remain to be overcome.

1.9.2.2. Enzymes in thrombolytic treatment

Activation of the blood clotting mechanism during inflammation is part of the body's defense mechanism which requires therapeutic intervention. Under normal physiological conditions there is an equilibrium between blood coagulation (clotting) and fibrinolysis (the process of dissolving the clotted blood) [ 47 ]. Biocatalysts such as enzymes, ribozymes, pro-enzymes, activators and pro-activators are responsible for maintaining equilibrium between clot formation and fibrinolysis. Imbalances in the concentration of these bio-activators may disturb physiology. In the biological process of fibrogenesis, clot formation takes place due to the plasma protein (soluble fibrinogen), which is ultimately converted to insoluble fibrin by the enzyme thrombin. This process is dependent on the conversion of thrombin from prothrombin. This bio-conversion takes place after the cascade of enzymatic reactions which involved certain key biological compounds called clotting factors. A blood clot dissolving enzyme known as plasmin is present in the blood as the pro-enzyme plasminogen. During clot dissolution activators convert the plasminogen to plasmin. This biological process is well regulated by certain process such as vasoconstriction, formation of a fibrin and clot platelet aggregation [ 46 ].

As the body utilizes enzymes in conserving this key balance of homeostasis, in a similar way we can utilize enzymes to repair or restore the homeostatic balance once it is lost. Several reports have shown that one of the best approaches for treating such clinical conditions is the administration of enzymes capable of converting plasminogen to plasmin (the enzyme which dissolves the clot) via intraveneous injection. This type of treatment is called therapeutic thrombolysis or thrombolytic therapy. In this treatment, pharmacological agents are used to medically induce clot breakdown [ 47 ]. Various novel thrombolytic agents have been derived from different sources for therapeutic use, such as from bacteria (streptokinase), the venom of the Malayan pit viper (Arvin), a filamentous fungus Koji mold Aspergillus oryzae (brinase), a South American snake (reptilase) and human urine (urokinase) [ 47 ].

Current advancements in thrombolytic therapy are more focused on the treatment of occlusions (blockages) of blood vessels. These types of therapy can be considered as life-saving and emergency medicine for life-threatening conditions such as myocardial infarction and massive pulmonary embolism, which are the most common reasons for cardiac arrest. This life-saving treatment is more reliable in preventing the blockages of vessels in the lungs and heart. Artery blockage conditions such as pulmonary embolism in the lungs by the formation of a clot creates tension on the right side of the heart, resulting in shortness of breath and chest pain mainly upon breathing in. Enzyme-based thrombolysis for treating massive pulmonary embolism has been considered as an effective approach to dissolving clots in these large vessels. Since surgical removal raises the chances of new blood clot formation that can cause another pulmonary embolism at the same or a different site, it is considered a dangerous practice and thrombolytic therapy is considered the more effective treatment [ 47 ]. Nevertheless, reoccurrence of clot formation or clot re-formation is very common in patients who have undergone enzyme-based thrombolytic treatment. Researchers from various organizations (1971) determined the effectiveness of streptokinase over heparin in reducing the chances of death in acute myocardial infarction patients. Significant results were obtained during this experiment. As discussed above, re-formation of the clot is one of the major concerns in fibrinolytic therapy. Most clinicians start treatment with a high dose of fibrinolytic agents, which is reduced later on. This approach may reduce disease progression for some time, but often increases the chances of clot re-formation. Even after the dissolution of the clot it is very difficult to maintain the same physiologically balanced environment (homeostasis) at the site of damaged tissues and the chance of new clot formation at that particular location is very high. Therefore, fibrinolytic based treatment is always accompanied by anticoagulants, such as heparin [ 46 ].

Major concerns associated with streptokinase therapy are fever, a tendency for bleeding, antigenicity (as with any foreign protein) and the difficulty of determining the proper dose [ 47 ]. Post-enzymatic treatment bleeding is one of the major concerns and it is also a concern when anticoagulants are used alone. According to current research, urokinase (produced in the kidneys and obtained from human urine) is considered safer than streptokinase. For the production of urokinase, 2300 l of urine is required to yield only 29 mg of purified urokinase, thus considering the expense involved in its manufacture, its clinical utilization has been restricted. Other examples are Arvin and reptilase. Utilization of these has been restricted for several reasons, but they are still considered as potential replacements for heparin as anticoagulants. Some researchers have noticed that optimum dose plays an important role and is one of the key factors in determining re-clot formation. Thorough investigation is required to overcome any shortcomings and increase the acceptance of these enzymes in therapeutic use [ 47 ].

1.9.2.3. The role of enzymes in digestive disorders and inflammations

Enzymes play an essential role in the management of various digestive disorders, such as exocrine pancreatic insufficiency [ 48 ]. Supplementation with enzymes may also be advantageous for other conditions associated with poor digestion, such as lactose intolerance. Generally, pancreatic enzymes such as porcine and bovine have been the preferred form of supplementation for exocrine pancreatic insufficiency [ 48 ]. Utilization of microbe-derived lipase has presented promise with reports showing benefits alike to pancreatic enzymes, but with a lower dosage concentration and a broader pH range. The safety and efficacy of enzymes derived from microbial species in the treatment of conditions such as malabsorption and lactose intolerance is promising. Plant-derived enzymes, e.g. bromelain from pineapple, serve as active digestive aids in the breakdown of proteins. Synergistic properties have also been reported using a combination of animal-based enzymes and microbe-derived enzymes or bromelain. Buccal administration of pancreatin (derived from an alcoholic extract of animal pancreas) enhances the enzymatic digestion of starch and proteins in patients with pancreatic cysts and pancreatitis. Pancreatin in combination with lipase is used to treat patients with fatty stools. Hydrolytic enzymes such as papain and fungal extracts ( Aspergillus niger and Aspergillus otyzae ) are used to enhance absorption from the small intestine [ 49 ]. These fungal extracts comprise amylases and proteases along with cellulases, which support the breakdown of the otherwise indigestible fibers of cabbages, etc, and thus reduce dyspepsia and flatulence [ 50 ]. Currently, micro-organisms are used at a large scale for the production of therapeutic enzymes. Among various micro-organisms Saccharomyces cerevisiae, Saccharomyces fragilis, Bacillus subtilis and two Aspergillus species are considered safe by the FDA (USA) for obtaining oral β -galactosidase (from A. oryzae ) which is often used by patients suffering from inherited intestinal disease lactose deficiency [ 51 ]. Children with this genetic disorder children are incapable of digesting milk lactose. Enzymatic preparations such as β -galactosidase catalyze the conversion of lactose to glucose and galactose, which are quickly absorbed by the intestine. Other enzymatic preparations, e.g. penicillinase (from B. subtilis ) are often used to treat hypersensitivity reactions caused by the antibiotic penicillin [ 52 ]. This enzyme catalyzes the conversion of penicillin to penicillanic acid, which is non-immunogenic. In addition, microbial and plant hydrolases are also used to decrease inflammation and edema [ 53 ]. Thrombin, trypsin, chymotrypsin, papain, streptokinase, streptodornase and sempeptidase are under clinical trial investigation. These enzymatic preparations are administered orally and have considerable proteolytic activity in the serum. Streptodornase has also displayed pain-relieving action on systemic injection [ 54 ]. Preparations have also been used to clean dirty wounds and necrotic tissue and to remove debris from second and third degree burns.

1.10. Plants and algae enzyme systems

Plant based foods are usually consumed in their raw form [ 68 ]. This eases the main concern with animal-based enzymes by preserving the integrity of the enzymes themselves. Moreover, plant-based digestive enzymes are effective over a broad scope of pH levels. This range is usually between 3.0 and 9.0, which is highly well-matched with the human gastrointestinal environment [ 55 – 72 ]. Thus plant-based enzymes are compatible for supporting comprehensive digestive health. Protease, amylase, lipase and cellulose are the important enzymes and are present in plants. Protease breaks down protein that can be present in meat, fish, poultry, eggs, cheese and nuts. Amylase assists your body with the breakdown and subsequent absorption of carbohydrates and starches. Lipase aids the digestion of fat. When your diet includes lipase-rich foods, it eases the production burden on the gall bladder, liver and pancreas. Cellulase is present in many fruits and vegetables, and it breaks down food fibers, which increases their nutritional value to our bodies. The presence of cellulase in plant-based sources is important, because it is not naturally present in the human body. Fruits and vegetables are an ideal source for enzymes. They are enzyme-rich and easily consumed without needing to be cooked or processed, ultimately preserving the full functionality of the enzymes. By using plant biotechnology several enzymes can be produced from plants as well algal resources [ 56 – 72 ].

During algal photosynthesis various proteins and enzymes are produced which can be utilized in economic development and environment management, such as in wastewater treatment, production of fine chemicals, and biodiesel production [ 56 – 72 ]. Due to their potential to capture and fix carbon dioxide using solar energy, photosynthetic marine algae are considered as potential models for the production of proteins. It has been recently observed that algal chloroplasts can be transformed for the production recombinant proteins [ 55 ]. Five different classes of recombinant enzymes; xylanase, α-galactosidase, phytase, phosphate anhydrolase, and β-mannanase, D. tertiolecta or C. reinhardtii were in the plastids of D. tertiolecta or C. reinhardtii. Similar strategies should allow for recombinant protein production in many species of marine algae [ 55 ].

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Any substance that speeds up a biochemical reaction without being a reactant is called a catalyst. The catalysts for biochemical reactions in living systems are known as enzymes. They are thus known as biological catalysts or biocatalysts.

What are Enzymes

Enzymes are protein macromolecules that are necessary to initiate or speed up the rate of chemical reactions in the bodies of living organisms. The molecules on which enzymes act are called substrates, and the substance formed is called the product.

They are found in all living cells that vary in type based on the function it performs. Enzymes help in the process of digestion, blood clotting, and hormone production.

Are All Enzymes Proteins

Almost all known enzymes are proteins. Previously it was believed that all enzymes are chemically protein in nature. Then, certain nucleic acids known as ribozymes are also found to have catalytic properties. Students study protein-based enzymes when they learn about this group of organic biomolecules. The reason is that very little is known about ribozymes.

Proteins are composed of long amino acids chains that are held together by peptide bonds. Some enzymes are made up of only one chain of amino acids, while most others are made of multiple chains.

Some enzymes require a non-protein part for their functioning, known as cofactors. Cofactors are essential for the functioning of the enzyme. An enzyme devoid of a cofactor is an apoenzyme, while an enzyme with its cofactor is called a holoenzyme.

There are three types of cofactors:

1) Prosthetic Groups : They remain tightly bound to an enzyme all the time. Example – FAD

2) Coenzyme : They bind to an enzyme only during catalysis. Example – NAD +

3) Metal Ions : Certain enzymes require a metal ion at their active site for catalysis. They form a coordinate bond with the enzyme. Example – Zn 2+

Structure: What are They Made of

Each enzyme is made up of a unique chain of amino acids and has a unique shape. It can assume any of the three types of structure – primary, secondary, and tertiary structure. Enzymes made of amino acids that are arranged in a polypeptide chain produce the  primary structure . The formed amino acid chain is called a polypeptide.

The protein folds upon itself when the hydrogen in the (NH 2 ) group and the oxygen in the (COOH) group forms a hydrogen bond. It results in the folding of the protein in the 2-dimensional plane. There are two types of  secondary structures  – α-helix and β-sheet.

As a result of folding the 2-D linear chain in the secondary structure, the protein can fold up further, which helps it gain a 3-D structure. This formation is the tertiary structure of the protein.

• Protein in nature except for ribozyme
• Activity depends on several environmental factors such as temperature, pH, and concentration of substrate and products
• Initiate chemical reaction(s)
• Accelerate the rate of chemical reaction(s). The rate of an enzyme-catalyzed reaction usually is (103 – 108) times faster than an uncatalyzed reaction.

Apart from the general properties, enzymes have four more essential properties. They are given below:

Catalytic 

• Have excellent catalytic power
• Highly capable; a small quantity of an enzyme can catalyze a large quantity of a specific substrates.
• It remains unaltered at the end of the reaction
• The turnover number ranges between 0.5 to 600000

Specificity

• Specific to a particular type of reaction. A particular enzyme catalyzes a particular reaction binding to a particular substrate.

Reversibility

• Most reactions are reversible. The reversibility depends on the requirement of the cell
• In some cases, there are separate enzymes for forwarding and backward reaction
• Some reactions are not reversible

Sensitivity to Temperature and pH

• Highly sensitive to heat, temperature, and pH
• Activity is maximum within a specific range of temperature and pH ranges. Below which they have reduced activity and above which they get denatured and thus lose their activity.

When and How does an Enzyme Work to Catalyze Reactions

Enzymes work by lowering the activation energy – the amount of energy needed for the reaction to begin. Enzymes work by binding to reactant molecules, holding them so that the formation and breaking of bonds during the process can take place readily.

Enzymes do not change the free energy of the reactants and the products and thus do not affect a reaction’s ∆G value. Instead, they work by lowering the transition state, an intermediate state in the reaction. They also keep the equilibrium constant (K eq ) same throughout the reaction.

While catalyzing a chemical reaction, an enzyme binds to one or more reactant molecules. These molecules are enzymes substrates. In some reactions, one substrate molecule breaks to form multiple products. In contrast, in others, two substrate molecules join to form one large molecule. The part of the enzyme where the specific substrate binds is called the active site . The basic mechanism is divided into two steps, as shown below:

Enzyme (E) + Substrate (S) <—–> Enzyme-Substrate complex (ES)

Enzyme-Substrate complex (ES) <—–> Enzyme ( E) + Product (P)

The above two steps can be combined as follows to give the complete reaction.

Overall Reaction:

Enzyme (E) + Substrate (S) <—–> Enzyme-Substrate complex (ES) <—–> Enzyme ( E) + Product (P)

The amino acids present in the active site of the enzyme give the enzyme a definite shape. The shape uniquely determines its substrate and helps it to bind and form the enzyme-substrate complex. The enzyme then converts the bound substrate to a product with itself remaining chemically unchanged.

How do They Speed up Reactions

As stated above, enzymes work by decreasing the activation energy, like all catalysts. They generally lower the activation energy by reducing the energy needed for reactants to react during a reaction. Lesser the activation energy of a reaction, the faster the rate of the reaction. They do so in the following ways:

• Bring the reactants together such that they do not need to expend energy moving about to collide.
• Position the reactants correctly so that they do not have to overcome intermolecular forces that would typically push them apart
• Change the pathway so that the reaction can occur by the pathway with lower activation energy.

What Happens to an Enzyme after a Biochemical Reaction

After the reaction, the products formed are released from the active site of the enzyme. The enzyme remains unaltered at the end of the reaction and thus is free to bind another substrate and catalyze a new reaction.  

Factors Affecting Enzyme Activity

The active site of the enzyme is sensitive to some environmental factors. The following factors regulate them:

• Temperature : Increasing temperature increases the reaction rate, and lowering a temperature slows down a reaction. However, extremely high temperatures can cause the enzyme to denature and lose its functional activity. There is a specific temperature at which the enzyme activity is at its greatest. The optimum temperature is around 35.5°C for the enzymes in human cells.
• pH : Each enzyme works best within a particular range of pH. Beyond that, it is found to have reduced activity. An extremely high temperature can cause the enzyme to denature
• Enzyme Concentration : Increasing enzyme concentration speeds up a reaction until there are available substrates to bind. Once all the substrates get bound to their enzymes, the reaction shall no longer increase.  
• Substrate Concentration : Increasing substrate concentration also increases the reaction rate to a certain point. Once all the enzymes get bound to their substrates, a further increase in the substrate concentration will not affect the reaction rate.

Apart from the above factors, enzyme activity is also affected by ion concentration and the presence or absence of activators and inhibitors. Thus, enzymes work best within a specific range of temperature and pH ranges. 

Types and Examples

According to the International Union of Biochemists (IUB), enzymes are classified into six categories or functional classes. They are classified based on the type of reaction they catalyze. They are given below:

Functions: Why are Enzymes Important

Enzymes influence most of the biochemical reactions that take place in living organisms, including humans. Enzymes catalyze almost 4,000 such reactions, and the number is expected to be even higher.

Animals, including humans, have a vast number of enzymes working inside the human body. They are widely grouped into metabolic, digestive, and food enzymes.

• Metabolic enzymes  help produce energy and detoxify them by breaking down food particles consisting of protein, fat, and carbohydrates.

Examples are carboxylases, dehydrogenases, oxidoreductases, kinases, lyases, transferases, and many more.

• Digestive enzymes  catalyze reactions that break down macromolecules – carbohydrates, proteins, and fats into smaller molecules that help the body to produce energy.

Examples are amylase, lipase, maltase, peptidase, and protease.

• Food enzymes  are not naturally found in the body of living organisms, but we get their benefit from the food and their supplements.

Examples are cellulase, papain, actinidin, bromelain, and ficin.

Listed below are some more enzymes present in our body with their purpose:

• Lactase : Breaks down lactose, the complex milk sugar
• Pectinase : Breaks down pectin found in fruits and vegetables
• Catalase : Breaks down hydrogen peroxide into water and oxygen
• DNA polymerase : Synthesize DNA from deoxyribonucleotides
• Trypsin : Breaks down protein into amino acids
• Acetylcholinesterase : Breaks down the neurotransmitter acetylcholine

Enzymes are also needed in industry and household products. They are also commercially used to produce fermented products such as beer, wine, and cheese. In the clothing industry, enzymes play a role in reducing impurities in cotton.

Ans . Yes, enzymes can be reused.

Ans . Proteolytic enzymes break down protein.

Ans . Amylase breaks down carbohydrates in our bodies.

Ans . DNA helicase unzips DNA.

Ans . All known enzymes are catalysts, but not all catalysts are enzymes.

Ans . No, enzymes are not used up in a reaction.

Ans . Lipase breaks down fat.

Ans . Amylase breaks down starch.

Ans . No, hemoglobin is not an enzyme. It is a protein.

Ans . The pancreas produces enzymes that break down nutrients.

Ans . Enzymes are primarily organic and are bimolecular, while catalysts can also be inorganic compounds.

Ans . No, enzymes do not have the same shape as their substrates.

Ans . Each enzyme’s active site is specific for one particular substrate – similar to a lock with a specific key. Changing the enzyme’s shape will prevent it from binding to its substrate, thus affecting its function.

Ans . High temperature and extreme pH can denature enzymes.

Ans . Ribosomes are enzymes made in the cell.

Ans . Enzymes only work on their specific substrates because the enzyme’s active site can bind to only a specific substrate – similar to a lock that has a specific key.

Ans . No, enzymes speed up chemical reactions by lowering the activating energy.

Ans . Enzyme inhibitors modify the enzyme’s catalytic properties, thus slowing down the reaction rate and, in some cases, even stopping the reaction.

Ans . The substrate binds to the enzyme at its active site.

Ans . RuBisCO makes up the most abundant protein on earth.

Ans . Yes, enzymes are found in all cells.

Ans . There are approximately 1300 different enzymes found in the human body.

• Enzymes – Bio.libretexts.org
• Enzyme – Genome.gov
• Enzymes – Sciencedirect.com
• What Is an Enzyme Structure and Function – Thoughtco.com
• Enzyme Biochemistry – Thoughtco.com

Article was last reviewed on Thursday, November 11, 2021

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4.6 Enzymes

A substance that helps a chemical reaction to occur is a catalyst, and the special molecules that catalyze biochemical reactions are enzymes. Almost all enzymes are proteins, comprised of amino acid chains. Enzymes facilitate chemical reactions by binding to the reactant molecules, and holding them in such a way as to make the chemical bond-breaking and bond-forming processes take place more readily.

Enzyme Active Site and Substrate Specificity

The chemical reactants to which an enzyme binds are the enzyme’s  substrates . There may be one or more substrates, depending on the particular chemical reaction. In some reactions, a single-reactant substrate breaks down into multiple products. In others, two substrates may come together to create one larger molecule. Two reactants might also enter a reaction, both become modified, and leave the reaction as two products. The location within the enzyme where the substrate binds is the enzyme’s  active site . This is where the “action” happens. Since enzymes are proteins, there is a unique combination of amino acid residues (also side chains, or R groups) within the active site. Different properties characterize each residue. These can be large or small, weakly acidic or basic, hydrophilic or hydrophobic, positively or negatively charged, or neutral. The unique combination of amino acid residues, their positions, sequences, structures, and properties, creates a very specific chemical environment within the active site. This specific environment is suited to bind, albeit briefly, to a specific chemical substrate (or substrates). Due to this jigsaw puzzle-like match between an enzyme and its substrates (which adapts to find the best fit between the transition state and the active site), enzymes are known for their specificity. The “best fit” results from the shape and the amino acid functional group’s attraction to the substrate. There is a specifically matched enzyme for each substrate and, thus, for each chemical reaction; however, there is flexibility as well.

The fact that active sites are so perfectly suited to provide specific environmental conditions also means that they are subject to local environmental influences. It is true that increasing the environmental temperature generally increases reaction rates, enzyme-catalyzed or otherwise. However, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the active site in such a way that they are less well suited to bind substrates. High temperatures will eventually cause enzymes, like other biological molecules, to denature , a process that changes the substance’s natural properties. Likewise, the local environment’s pH can also affect enzyme function. Active site amino acid residues have their own acidic or basic properties that are optimal for catalysis. These residues are sensitive to changes in pH that can impair the way substrate molecules bind. Enzymes are suited to function best within a certain pH range, and, as with temperature, extreme environmental pH values (acidic or basic) can cause enzymes to denature.

Induced Fit and Enzyme Function

For many years, scientists thought that enzyme-substrate binding took place in a simple “lock-and-key” fashion. This model asserted that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call  induced fit  ( Figure 1 ). This model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild shift in the enzyme’s structure that confirms an ideal binding arrangement between the enzyme and the substrate’s transition state. This ideal binding maximizes the enzyme’s ability to catalyze its reaction.

Link to Learning

When an enzyme binds its substrate, it forms an enzyme-substrate complex. This complex promotes the reaction’s rapid progression in one of many ways. On a basic level, enzymes promote chemical reactions that involve more than one substrate by bringing the substrates together in an optimal orientation. The appropriate region (atoms and bonds) of one molecule is juxtaposed to the other molecule’s appropriate region with which it must react. Another way in which enzymes promote substrate reaction is by creating an optimal environment within the active site for the reaction to occur. Certain chemical reactions might proceed best in a slightly acidic or non-polar environment. The chemical properties that emerge from the particular arrangement of amino acid residues within an active site create the perfect environment for an enzyme’s specific substrates to react.

The enzyme-substrate complex can also facilitate reactions by contorting substrate molecules in such a way as to facilitate bond-breaking, helping to reach the reaction to proceed. Finally, enzymes can also facilitate reactions by taking part in the chemical reaction itself. The amino acid residues can provide certain ions or chemical groups that actually form covalent bonds with substrate molecules as a necessary step of the reaction process. In these cases, it is important to remember that the enzyme will always return to its original state at the reaction’s completion. One of enzymes’ hallmark properties is that they remain ultimately unchanged by the reactions they catalyze. After an enzyme catalyzes a reaction, it releases its product(s).

Metabolism Control Through Enzyme Regulation

It would seem ideal to have a scenario in which all the encoded enzymes in an organism’s genome existed in abundant supply and functioned optimally under all cellular conditions, in all cells, at all times. In reality, this is far from the case. A variety of mechanisms ensure that this does not happen. Cellular needs and conditions vary from cell to cell, and change within individual cells over time. The required enzymes and energetic demands of stomach cells are different from those of fat storage cells, skin cells, blood cells, and nerve cells. Furthermore, a digestive cell works much harder to process and break down nutrients during the time that closely follows a meal compared with many hours after a meal. As these cellular demands and conditions vary, so do the amounts and functionality of different enzymes.

The relative amounts and functioning of the variety of enzymes within a cell ultimately determine which reactions will proceed and at which rates in that cell. This determination is tightly controlled. In certain cellular environments, environmental factors like pH and temperature partly control enzyme activity. There are other mechanisms through which cells control enzyme activity and determine the rates at which various biochemical reactions will occur.

Molecular Regulation of Enzymes

Enzymes can be regulated in ways that either promote or reduce their activity. There are many different kinds of molecules that inhibit or promote enzyme function, and various mechanisms exist for doing so. For example, in some cases of enzyme inhibition, an inhibitor molecule is similar enough to a substrate that it can bind to the active site and simply block the substrate from binding. Alternatively, in noncompetitive inhibition, an inhibitor molecule binds to the enzyme in a location other than an active site, a binding site away from the active site, and still manages to block substrate binding to the active site.

Drug Discovery by Looking for Inhibitors of Key Enzymes in Specific Pathways

Enzymes are key components of metabolic pathways. Understanding how enzymes work and how they can be regulated is a key principle behind developing many pharmaceutical drugs ( Figure 2 ) on the market today. Biologists working in this field collaborate with other scientists, usually chemists, to design drugs.

Consider statins for example—which is a class of drugs that reduces cholesterol levels. These compounds are essentially inhibitors of the enzyme HMG-CoA reductase. HMG-CoA reductase is the enzyme that synthesizes cholesterol from lipids in the body. By inhibiting this enzyme, the drug reduces cholesterol levels synthesized in the body. Similarly, acetaminophen, popularly marketed under the brand name Tylenol, is an inhibitor of the enzyme cyclooxygenase. While it is effective in providing relief from fever and inflammation (pain), scientists still do not completely understand its mechanism of action.

How are drugs developed? One of the first challenges in drug development is identifying the specific molecule that the drug is intended to target. In the case of statins, HMG-CoA reductase is the drug target. Researchers identify targets through painstaking research in the laboratory. Identifying the target alone is not sufficient. Scientists also need to know how the target acts inside the cell and which reactions go awry in the case of disease. Once researchers identify the target and the pathway, then the actual drug design process begins. During this stage, chemists and biologists work together to design and synthesize molecules that can either block or activate a particular reaction. However, this is only the beginning: both if and when a drug prototype is successful in performing its function, then it must undergo many tests from  in vitro  experiments to clinical trials before it can obtain FDA approval to be on the market.

Many enzymes don’t work optimally, or even at all, unless bound to other specific non-protein helper molecules, either temporarily through ionic or hydrogen bonds or permanently through stronger covalent bonds. Two types of helper molecules are  cofactors  and  coenzymes . Binding to these molecules promotes optimal conformation and function for their respective enzymes. Cofactors are inorganic ions such as iron (Fe++) and magnesium (Mg++). One example of an enzyme that requires a metal ion as a cofactor is the enzyme that builds DNA molecules, DNA polymerase, which requires a bound zinc ion (Zn++) to function. Coenzymes are organic helper molecules, with a basic atomic structure comprised of carbon and hydrogen, which are required for enzyme action. The most common sources of coenzymes are dietary vitamins ( Figure 3 ). Some vitamins are precursors to coenzymes and others act directly as coenzymes. Vitamin C is a coenzyme for multiple enzymes that take part in building the important connective tissue component, collagen. An important step in breaking down glucose to yield energy is catalysis by a multi-enzyme complex scientists call pyruvate dehydrogenase. Pyruvate dehydrogenase is a complex of several enzymes that actually requires one cofactor (a magnesium ion) and five different organic coenzymes to catalyze its specific chemical reaction. Therefore, enzyme function is, in part, regulated by an abundance of various cofactors and coenzymes, which the diets of most organisms supply.

Enzyme Compartmentalization

In animal cells, molecules such as enzymes are usually compartmentalized into different organelles. This allows for yet another level of regulation of enzyme activity. Enzymes required only for certain cellular processes are sometimes housed separately along with their substrates, allowing for more efficient chemical reactions. Examples of this sort of enzyme regulation based on location and proximity include the enzymes involved in the latter stages of cellular respiration, which take place exclusively in the mitochondria, and the enzymes involved in digesting cellular debris and foreign materials, located within lysosomes.

Section Summary

Enzymes are chemical catalysts that accelerate chemical reactions at physiological temperatures by lowering their activation energy. Enzymes are usually proteins consisting of one or more polypeptide chains. Enzymes have an active site that provides a unique chemical environment, comprised of certain amino acid R groups (residues). This unique environment is perfectly suited to convert particular chemical reactants for that enzyme, scientists call substrates, into unstable intermediates that they call transition states. Enzymes and substrates bind with an induced fit, which means that enzymes undergo slight conformational adjustments upon substrate contact, leading to full, optimal binding. Enzymes bind to substrates and catalyze reactions in four different ways: bringing substrates together in an optimal orientation, compromising the bond structures of substrates so that bonds can break down more easily, providing optimal environmental conditions for a reaction to occur, or participating directly in their chemical reaction by forming transient covalent bonds with the substrates.

Enzyme action must be regulated so that in a given cell at a given time, the desired reactions catalyze and the undesired reactions are not. Enzymes are regulated by cellular conditions, such as temperature and pH. They are also regulated through their location within a cell, sometimes compartmentalized so that they can only catalyze reactions under certain circumstances. Enzyme inhibition and activation via other molecules are other important ways that enzymes are regulated.

Human Biology Copyright © by Sarah Malmquist and Kristina Prescott is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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Specificity of Enzyme

Introduction.

The cell is the fundamental unit of life which serves as both its structural and functional foundation. Cells can use biocatalysts, referred to as enzymes, which have excellent catalytic efficiency, both substrate and reaction specificity. Enzymes are ideal for biological reactions due to their extraordinary catalytic power and high level of substrate specificity. They are essential for the metabolism of cells.

The complex protein molecules produced by living cells are known as enzymes or biocatalysts. Both the processes they catalyse and the reactants they choose, or substrates, are highly selective. An enzyme normally catalyses one chemical reaction or a group of related reactions.

Enzymes’ specificity concerning the reactions they catalyse is one of their characteristics that makes them so significant as diagnostic and research tools. A small number of enzymes exhibit the ability to catalyse only one specific reaction. Other enzymes will have a preference for a certain kind of functional group or chemical bond.

Table of Contents

Enzyme specificity, types of enzyme specificity.

• Frequently Asked Questions (FAQs)

An enzyme is a component that functions as a catalyst in living beings, controlling the frequency at which chemical reactions occur without changing the enzyme itself.

All biological activities in living organisms involve chemical reactions, and enzymes regulate most of them. Many of these activities would not occur at a significant rate without enzymes. Enzymes catalyse every aspect of cell metabolism.

Enzymes are useful in industrial and medical applications. Wine fermentation, bread leavening, cheese curdling, and beer brewing have all been done since the beginning of time, but not till the 19th century that it was realised that these events were the result of enzymes’ catalytic activity. Since then, enzymes have become more significant in industry applications involving organic chemical reactions. Enzymes are used in medicine for various purposes, including aiding wound healing, identifying diseases, and eliminating disease-causing microbes.

An enzyme has four primary features.

• They are very catalytic and can easily catalyse a chemical reaction.
• They enhance reactions while remaining consistent throughout.
• Enzyme effectiveness and function are easily affected by temperature, pH, and inhibitors.
• Enzymes seem to be quite specific and mainly catalyse only one type of substrate.

Mechanism of Enzyme Actions

An enzyme pulls substrates to its active site, catalyses the chemical reaction that produces the products, and then enables the dissociation of the products (detach from the enzyme surface). The enzyme and substrate complex is the interaction between an enzyme and its substrates.

One substrate and one enzyme constitute a binary complex, whereas two substrates and one enzyme constitute a ternary complex. The substrates are drawn towards the active site by hydrophobic and electrostatic forces, considered noncovalent bonds since they possess physical attractions and are not chemical bonds.

Enzymes possess an active site. The functional group that allows reactant molecules to attach to the active site is a specific shape-defined region of the molecule. The substrate group refers to the molecule that connects to the enzyme. Without the help of a catalyst, the substrate and the enzyme produce an intermediate reaction with low activation energy.

Reactant (1) + Reactant (2) → Product

Reactant (1) + Enzyme → Intermediate

Intermediate + Reactant (2) → Product + Enzyme

The key mechanism of enzymatic activity is to catalyse chemical reactions, which originates with the substrate’s binding to the enzyme’s active site. This active site is a specific area where the substrate interacts.

Also, read: Digestive Enzymes

The ability of an enzyme to select a specific substrate from a range of chemically similar compounds is known as specificity. Since the enzyme and substrate exhibit complementary structural and conformational properties, specificity is a molecular identification process. Different enzymes exhibit different levels of substrate specificity.

The specificity that enzymes show to the reactions they catalyse is one of the characteristics that makes them so useful as diagnostic and research tools. Only a selected few enzymes can catalyse a single reaction or they have perfect specificity. Other enzymes will have a preference for a certain kind of functional group or chemical bond. There are usually four different categories of specificity:

• Absolute specificity – The enzyme catalyses only one reaction.
• Group specificity – The enzyme acts only on molecules having specific functional groups, like phosphate, amino, and methyl groups.
• Linkage specificity – The enzyme acts on a specific type of chemical bond regardless of the remaining molecular structure.
• Stereochemical specificity – The enzyme acts on a certain optical or steric isomer.

Even though enzymes have high levels of specificity, cofactors can be used by numerous apoenzymes. The reactions of lactate dehydrogenase, malate dehydrogenase, and alcohol dehydrogenase are a few among them. For instance, nicotinamide adenine dinucleotide (NAD) serves as a hydrogen acceptor in a large number of dehydrogenase activities and is a coenzyme for those reactions.

Enzyme Specificity Example

Enzymes vary in their degree of specificity. Due to the specificity of enzyme function, digestive enzymes like pepsin and chymotrypsin can interact with any protein. Thrombin is a component of a very delicate blood-clotting mechanism that can only respond with one substance. This is done to maintain the normal functioning of the system since it only interacts with the protein fibrinogen.

Oxidoreductases do not catalyse reactions involving hydrolysis, and hydrolases do not catalyse processes involving both oxidation and reduction. As a result, an enzyme can catalyse a specific chemical reaction and various substances that are similar to it.

Only suitably structured molecules may act as substrates for a specific enzyme because the substrate must fit into the active site of the enzyme preceding catalysis. An enzyme will often react with one naturally existing compound. The concept of enzyme specificity will be demonstrated using two oxidoreductase enzymes.

First, alcohol dehydrogenase (ADH) reacts with alcohol, and then lactate dehydrogenase (LDH) reacts with lactic acid. Despite being oxidoreductase enzymes, the two actions are not interchangeable. It indicates that alcohol dehydrogenase cannot catalyse a process involving lactic acid and vice versa. This is because each substrate has a specific structure that makes it impossible to fit into the active site of a different enzyme.

Enzyme specificity is significant since it recognises the various metabolic pathways consisting of a large number of enzymes.

Specificity is attained when a chemical reactant (the substrate) interacts weakly with an enzyme’s active site to form a bond. The production and dissolution of covalent bonds is the only sort of chemical reaction that an enzyme can catalyse. Reaction specificity, also referred to as absolute reaction specificity, is a property of enzymes that refers to their specificity to a single reaction, meaning that no by-products are produced.

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What are enzymes and what do they do in our bodies? Enzymes are basically proteins that are produced by living organisms to bring about certain metabolic and biochemical reactions in the body. They are biological catalysts that speed up reactions inside the body. Let’s find out more about them.

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What is the structure of enzymes.

Enzymes, as mentioned above, are biological catalysts. While they hasten or speed up a process, they are actually providing an alternative pathway for the process. But, in the process, the structure or composition of the enzymes remain unaltered.

Enzymes are actually made up of 1000s of amino acids that are linked in a  specific way to form different enzymes. The enzyme chains fold over to form unique shapes and it is these shapes that provide the enzyme with its characteristic chemical potential. Most enzymes also contain a non-protein component known as the co-facto r.   

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Types of Enzymes:

The biochemical reactions occurring in the body are basically of 6 types and the enzymes that bring about these reactions are named accordingly:

• Oxidoreductases: These enzymes bring about oxidation and reduction reactions and hence are called oxidoreductases. In these reactions, electrons in the form of hydride ions or hydrogen atoms are transferred. When a substrate is being oxidized, these enzymes act as the hydrogen donor. These enzymes are called dehydrogenases or reductases. When the oxygen atom is the acceptor, these enzymes are called oxidases.
• Transferases: These enzymes are responsible for transferring functional groups from one molecule to another. Example: alanine aminotransferase which shuffles the alpha‐amino group between alanine and aspartate etc. Some transferases also transfer phosphate groups between ATP and other compounds, sugar residues to form disaccharides such as hexokinase in glycolysis.
• Hydrolases: These enzymes catalyze reactions that involve the process of hydrolysis.They break single bonds by adding water. Some hydrolases function as digestive enzymes because they break the peptide bonds in proteins. Hydrolases can also be a type of transferases as they transfer the water molecule from one compound to another. Example: Glucose-6-phosphatase that removes the phosphate group from glucose-6-phosphate, leaving glucose and H 3 PO 4 .
• Lyases:   These enzymes catalyze reactions where functional groups are added to break double bonds in molecules or where double bonds are formed by the removal of functional groups. Example: Pyruvate decarboxylase is a lyase that removes CO 2  from pyruvate. Other examples include deaminases and dehydratases.
• Isomerases: These enzymes catalyze the reactions where a functional group is moved to another position within the same molecule such that the resulting molecule is actually an isomer of the earlier molecule. Example: triosephosphate isomerase and phosphoglucose isomerase for converting glucose 6-phosphate to fructose 6-phosphate.
• Ligases: These enzymes perform a function that is opposite to that of the hydrolases. Where hydrolases break bonds by adding water, ligases form bonds by removal of the water component. There are different subclasses of ligases which involve the synthesis of ATP.

How do enzymes work?

For any reaction to occur in the universe, there is an energy requirement. In cases where there is no activation energy provided, a catalyst plays an important role to reduce the activation energy and carried forward the reaction. This works in animals and plants as well. Enzymes help reduce the activation energy of the complex molecules in the reaction.  The following steps simplify how an enzyme works to speed up a reaction:

Step 1 : Each enzyme has an ‘active site’ which is where one of the substrate molecules can bind to. Thus, an enzyme- substrate complex is formed.

Step 2: This enzyme-substrate molecule now reacts with the second substrate to form the product and the enzyme is liberated as the second product.

There are many theories that explain how enzymes work. But, there are two important theories that we will discuss here.

Theory 1: Lock and Key Hypothesis

This is the most accepted of the theories of enzyme action.

This theory states that the substrate fits exactly into the active site of the enzyme to form an enzyme-substrate complex. This model also describes why enzymes are so specific in their action because they are specific to the substrate molecules.

Theory 2: Induced Fit Hypothesis

This is similar to the lock and key hypothesis. It says that the shape of the enzyme molecule changes as it gets closer to the substrate molecule in such a way that the substrate molecule fits exactly into the active site of the enzyme.

What factors affect enzyme activity in the cell?

• Concentration of Enzymes and Substrates: The rate of reaction increases with increasing substrate concentration up to a point, beyond which any further increase in substrate concentration produces no significant change in reaction rate. This occurs because after a certain concentration of the substrate, all the active sites on the enzyme are full and no further reaction can occur.
• Temperature: With the increase in temperature, the enzyme activity increases because of the increase in kinetic energy of the molecules. There is an optimum level when the enzymes work at the best and maximum. This temperature is often the normal body temperature of the body. When the temperature increases beyond a certain limit, enzymes, which are actually made up of proteins, begin to disintegrate and the rate of reaction slows down.
• pH: Enzymes are very sensitive to changes in the pH and work in a very small window of permissible pH levels. Below or above the optimum pH level, there is a risk of the enzymes disintegrating and thereby the reaction slows down.
• Inhibitors: Presence of certain substances that inhibit the action of a particular enzyme. This occurs when the inhibiting substance attaches itself to the active site of the enzyme thereby preventing the substrate attachment and slows down the process.

Solved Example for You

Q: An enzyme acts by?

a. Increasing the energy of activation

b. Decreasing the energy of activation

c. Decreasing the pH

d. Increasing the pH

Sol: a. Increasing the energy of activation

The reactants do not undergo chemical change automatically. They do so in the transition state. Transition state has more free energy than reactants or products. The inability of reactants to undergo change due to the requirement of extra energy for converting them to transition state is called as ‘Energy Barrier’. The energy required to overcome energy barrier is called as ‘Activation Energy’.

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Chemical nature

Nomenclature, mechanism of enzyme action.

• Factors affecting enzyme activity

What is an enzyme?

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• National Library of Medicine - Enzymes: principles and biotechnological applications
• Open Oregon Educational Resources - Enzymes
• Khan Academy - Enzyme structure and function
• Royal Society of Biology - Enzymes
• Royal Society of Chemistry Publishing - Role of conformational dynamics in the evolution of novel enzyme function
• Cleveland Clinic - Enzymes
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• enzyme - Children's Encyclopedia (Ages 8-11)
• enzyme - Student Encyclopedia (Ages 11 and up)
• Table Of Contents
• An enzyme is a substance that acts as a catalyst in living organisms, regulating the rate at which chemical reactions proceed without itself being altered in the process.
• The biological processes that occur within all living organisms are chemical reactions, and most are regulated by enzymes.
• Without enzymes, many of these reactions would not take place at a perceptible rate.
• Enzymes catalyze all aspects of cell metabolism. This includes the digestion of food, in which large nutrient molecules (such as proteins, carbohydrates, and fats) are broken down into smaller molecules; the conservation and transformation of chemical energy; and the construction of cellular macromolecules from smaller precursors.
• Many inherited human diseases, such as albinism and phenylketonuria , result from a deficiency of a particular enzyme.
• A large protein enzyme molecule is composed of one or more amino acid chains called polypeptide chains. The amino acid sequence determines the characteristic folding patterns of the protein’s structure, which is essential to enzyme specificity.
• If the enzyme is subjected to changes, such as fluctuations in temperature or pH, the protein structure may lose its integrity (denature) and its enzymatic ability.
• Bound to some enzymes is an additional chemical component called a cofactor , which is a direct participant in the catalytic event and thus is required for enzymatic activity. A cofactor may be either a coenzyme —an organic molecule, such as a vitamin—or an inorganic metal ion. Some enzymes require both.
• All enzymes were once thought to be proteins, but since the 1980s the catalytic ability of certain nucleic acids, called ribozymes (or catalytic RNAs), has been demonstrated, refuting this axiom.
• Practically all of the numerous and complex biochemical reactions that take place in animals, plants, and microorganisms are regulated by enzymes, and so there are many examples. Among some of the better-known enzymes are the digestive enzymes of animals. The enzyme pepsin , for example, is a critical component of gastric juices, helping to break down food particles in the stomach. Likewise, the enzyme amylase , which is present in saliva, converts starch into sugar, helping to initiate digestion.
• In medicine, the enzyme thrombin is used to promote wound healing. Other enzymes are used to diagnose certain diseases. The enzyme lysozyme , which destroys cell walls, is used to kill bacteria.
• The enzyme catalase brings about the reaction by which hydrogen peroxide is decomposed to water and oxygen. Catalase protects cellular organelles and tissues from damage by peroxide, which is continuously produced by metabolic reactions.
• Enzyme activity is affected by various factors, including substrate concentration and the presence of inhibiting molecules.
• The rate of an enzymatic reaction increases with increased substrate concentration, reaching maximum velocity when all active sites of the enzyme molecules are engaged. Thus, enzymatic reaction rate is determined by the speed at which the active sites convert substrate to product.
• Inhibition of enzyme activity occurs in different ways. Competitive inhibition occurs when molecules similar to the substrate molecules bind to the active site and prevent binding of the actual substrate.
• Noncompetitive inhibition occurs when an inhibitor binds to the enzyme at a location other than the active site.
• Another factor affecting enzyme activity is allosteric control , which can involve stimulation of enzyme action as well as inhibition. Allosteric stimulation and inhibition allow production of energy and materials by the cell when they are needed and inhibit production when the supply is adequate.

enzyme , a substance that acts as a catalyst in living organisms, regulating the rate at which chemical reactions proceed without itself being altered in the process.

A brief treatment of enzymes follows. For full treatment, see protein: Enzymes .

The biological processes that occur within all living organisms are chemical reactions , and most are regulated by enzymes. Without enzymes, many of these reactions would not take place at a perceptible rate. Enzymes catalyze all aspects of cell metabolism . This includes the digestion of food, in which large nutrient molecules (such as proteins , carbohydrates , and fats ) are broken down into smaller molecules; the conservation and transformation of chemical energy ; and the construction of cellular macromolecules from smaller precursors . Many inherited human diseases, such as albinism and phenylketonuria , result from a deficiency of a particular enzyme.

Enzymes also have valuable industrial and medical applications. The fermenting of wine, leavening of bread, curdling of cheese , and brewing of beer have been practiced from earliest times, but not until the 19th century were these reactions understood to be the result of the catalytic activity of enzymes. Since then, enzymes have assumed an increasing importance in industrial processes that involve organic chemical reactions. The uses of enzymes in medicine include killing disease-causing microorganisms, promoting wound healing, and diagnosing certain diseases.

All enzymes were once thought to be proteins, but since the 1980s the catalytic ability of certain nucleic acids, called ribozymes (or catalytic RNAs), has been demonstrated, refuting this axiom. Because so little is yet known about the enzymatic functioning of RNA , this discussion will focus primarily on protein enzymes.

A large protein enzyme molecule is composed of one or more amino acid chains called polypeptide chains. The amino acid sequence determines the characteristic folding patterns of the protein’s structure, which is essential to enzyme specificity. If the enzyme is subjected to changes, such as fluctuations in temperature or pH, the protein structure may lose its integrity (denature) and its enzymatic ability. Denaturation is sometimes, but not always, reversible.

Bound to some enzymes is an additional chemical component called a cofactor , which is a direct participant in the catalytic event and thus is required for enzymatic activity. A cofactor may be either a coenzyme —an organic molecule, such as a vitamin —or an inorganic metal ion ; some enzymes require both. A cofactor may be either tightly or loosely bound to the enzyme. If tightly connected, the cofactor is referred to as a prosthetic group.

An enzyme will interact with only one type of substance or group of substances, called the substrate , to catalyze a certain kind of reaction. Because of this specificity, enzymes often have been named by adding the suffix “-ase” to the substrate’s name (as in urease , which catalyzes the breakdown of urea ). Not all enzymes have been named in this manner, however, and to ease the confusion surrounding enzyme nomenclature , a classification system has been developed based on the type of reaction the enzyme catalyzes. There are six principal categories and their reactions: (1) oxidoreductases , which are involved in electron transfer; (2) transferases , which transfer a chemical group from one substance to another; (3) hydrolases , which cleave the substrate by uptake of a water molecule (hydrolysis); (4) lyases , which form double bonds by adding or removing a chemical group; (5) isomerases , which transfer a group within a molecule to form an isomer; and (6) ligases , or synthetases, which couple the formation of various chemical bonds to the breakdown of a pyrophosphate bond in adenosine triphosphate or a similar nucleotide .

In most chemical reactions, an energy barrier exists that must be overcome for the reaction to occur. This barrier prevents complex molecules such as proteins and nucleic acids from spontaneously degrading, and so is necessary for the preservation of life. When metabolic changes are required in a cell, however, certain of these complex molecules must be broken down, and this energy barrier must be surmounted. Heat could provide the additional needed energy (called activation energy ), but the rise in temperature would kill the cell. The alternative is to lower the activation energy level through the use of a catalyst . This is the role that enzymes play. They react with the substrate to form an intermediate complex—a “transition state”—that requires less energy for the reaction to proceed. The unstable intermediate compound quickly breaks down to form reaction products, and the unchanged enzyme is free to react with other substrate molecules.

Only a certain region of the enzyme, called the active site , binds to the substrate. The active site is a groove or pocket formed by the folding pattern of the protein. This three-dimensional structure, together with the chemical and electrical properties of the amino acids and cofactors within the active site, permits only a particular substrate to bind to the site, thus determining the enzyme’s specificity.

Enzyme synthesis and activity also are influenced by genetic control and distribution in a cell. Some enzymes are not produced by certain cells, and others are formed only when required. Enzymes are not always found uniformly within a cell; often they are compartmentalized in the nucleus , on the cell membrane , or in subcellular structures. The rates of enzyme synthesis and activity are further influenced by hormones , neurosecretions, and other chemicals that affect the cell’s internal environment .