Chapter 3

Enzymology
Theresa May Chin Tan Department of Biochemistry, National University of Singapore 8 Medical Drive, Singapore 117597 Email: [email protected]

3.1. Enzymes as Catalysts Life depends on a series of coordinated chemical reactions. Many of these reactions however proceed too slowly to sustain life and hence proteins with catalytic functions have evolved. An enzyme is a protein that functions as a catalyst. The role of an enzyme (or a catalyst) is to speed up the rate of chemical reactions without undergoing any permanent changes itself. At the end of the reaction, the enzyme remains unchanged. The rate of a reaction in the presence of an enzyme is between 106 to 1014 fold higher than the uncatalyzed reaction. How is this achieved? In any chemical reaction, a reactant must contort into an unfavorable highenergy conformation before it changes into the product. The formation of this less favorable transition state represents an activation energy barrier that must be overcome before the reaction can occur. Enzymes act to lower the activation energy barrier and therefore facilitate the progress of the reaction (Fig. 3.1). Enzyme-catalyzed reactions can thus proceed at a greater rate. Enzymes are protein molecules. Extreme conditions such as high temperature or extreme pH will affect the structure of an enzyme. This will in turn affect the catalytic activity. Most human enzymes have an optimal
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temperature around 37C. At low temperatures, the rate of an enzymatic reaction is reduced. At high temperatures, denaturation of the enzyme occurs and loss of activity is observed. For most enzymes, an increase in reaction rate is usually observed as the pH goes from an acidic level toward physiological range and then declines as pH progresses towards the alkaline range. Hence, unlike chemical catalysts, enzymes catalyze reactions under relatively mild conditions: moderate temperature, atmospheric pressure and nearly neutral pH. In addition, the catalytic properties of many enzymes can be regulated within the living organism. Each enzymatic reaction proceeds with great specificity. How do enzymes achieve specificity? An enzyme will bind to the substrate (reactant); form an enzyme-substrate complex and covert the substrate into the product. This occurs in a relative small portion of the enzyme called the active (or catalytic) site. The specificity of the enzymatic reaction results from the three-dimensional arrangement of specific amino-acids residues in the enzyme that forms the substrate-binding site. Two models have been used to describe enzyme-substrate interaction. In the lock and key model, the enzyme is viewed as the lock and the substrate as the key. Only one

Free energy, G

Uncatalyzed

Free energy of activation

Catalyzed

A P

Net energy change

Reaction coordinate
Fig. 3.1. Transition state diagram of an uncatalyzed and a catalyzed reaction. The net free energy change is the same for both but the free energy of activation is lowered in the presence of a catalyst. Hence, in the presence of a catalyst, it is easier for the reactant, A, to reach the transition state, T, and be converted to the product, P.

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Enzymology 75

Substrate (key)

Enzyme (lock)

Fig. 3.2. Lock and key model for enzyme-substrate interaction. The enzyme is analogous to a lock while the substrate is viewed as the key that will fit specifically into the lock.

Substrate

Enzyme

Fig. 3.3. Induced fit model of enzyme-substrate interaction. Substrate binding will alter the conformation of the active site such that the enzyme and substrate will fit each other more precisely.

key will fit each lock (Fig. 3.2). This model presents the active site as a rigid unchanging structure. In the induced fit model, the enzyme is not presented as a rigid molecule. Rather, it can change its conformation as the substrate binds and as the reaction proceeds (Fig. 3.3). Conformational changes have indeed been proven to occur by X-ray crystallographic studies on the yeast hexokinase enzyme. 3.2. Nomenclature Before the Enzyme Commission (EC) recommended the present system of nomenclature, enzymes were given trivial names. The trivial names consisted of the suffix -ase added to the substrate of the enzyme (e.g. urease), or implied something about the nature of the reaction

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(e.g. alcohol dehydrogenase), or simply did not give any clue to what the enzyme does (e.g. trypsin). The EC was set up by the International Union of Biochemistry (now known as the International Union of Biochemistry and Molecular Biology IUBMB). Based on the EC recommendation, enzymes are classified and named according to the nature of the chemical reactions they catalyzed. There are six major classes of enzyme reactions (Table 3.1). The major classes are further subdivided into subclasses and sub-subclasses. Each enzyme has a unique EC number and a systematic name with the suffix -ase. The systematic name of an enzyme consists of the name of its substrate(s) followed by a word ending with ase specifying the type of reaction the enzyme catalyses. The enzyme, glucokinase, catalyzes the transfer of a phosphate from ATP to D-glucose. Its EC number is 2.7.1.2 and its systematic name is ATP:D-glucose 6-phosphotransferase. The number represents the class, subclass, sub-subclass and its arbitrarily assigned serial number in the sub-subclass. The ENZYME database (http://tw.expasy.org/enzyme/) is a repository of information on the nomenclature of enzymes. It contains the following data for each enzyme: EC number, recommended name, alternative names, catalytic activity, cofactors, links to the SWISS-PROT protein sequence entries that correspond to the enzyme and links to human diseases associated with a deficiency of the enzyme.

Table 3.1. Classification of enzyme reactions. EC number 1 2 3 4 5 6 Type of reaction Oxidation-reduction reactions Transfer of functional groups Hydrolysis reactions Addition of groups to double bonds or formation of double bonds Isomerization reactions Bond formation coupled with hydrolysis of high energy compounds such as ATP Class of enzyme Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases

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Enzymology 77

3.3. Cofactors Enzymes have evolved to cope with the necessity of catalyzing chemical reactions within a living cell. To improve the repertoire of reactions which enzymes are able to catalyze, cofactors are often necessary. Without cofactors, the functional groups present on the side chains of amino acids on enzymes can only facilitate acid-base reactions, form certain types of transient covalent bonds and take part in charge-charge interactions. The presence of cofactors provides additional functional groups and allows enzymes to catalyze other types of reactions (e.g. oxidation-reduction reactions). Thus, enzymes can be divided into 2 groups: those that rely solely on their protein structure to carry out their catalytic function and those that require an additional cofactor to carry out the catalytic activity. Cofactors may be metal ions (such as Zn++, Fe++) or organic molecules. Such organic molecules are referred to as coenzymes and are derived from vitamins. Flavin coenzymes (flavin adenine dinucleotide, FAD and flavin mononucleotide, FMN) are examples of coenzymes and the flavin component is derived from the vitamin riboflavin (also known as vitamin B2). Some cofactors are transiently associated with the given enzyme while others are permanently associated with their protein. The latter are called prosthetic groups. The catalytic active complex of enzyme-prosthetic group is called the holoenzyme. Without the prosthetic group, the protein portion is inactive and is referred to as the apoenzyme. 3.4. Regulation of Enzyme Activity The catalytic activities of many enzymes are regulated in a living cell. Regulation can be achieved in many different ways. The different modes of regulation include: allosteric control, regulatory proteins, covalent modification of enzymes, synthesis of inactive precursors and control of enzyme synthesis. (1) Control of Enzyme Synthesis Bacteria can rapidly adapt to their environment by producing enzymes that can metabolize available nutrients. An example is the induction of lactose metabolizing enzymes, -galactosidase and galactose permease.

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Bacteria grown in the absence of lactose express little of these enzymes. In the presence of lactose, the synthesis of -galactosidase and galactose permease is rapidly induced and the bacteria can now utilize lactose as a nutrient source. The enzymes are expressed only when required and regulation is achieved by controlling gene expression. (2) Inactive Precursors Regulation of enzyme activity can also be achieved by synthesizing enzymes as inactive precursors. The inactive precursors are known as zymogens or proenzymes. Digestive enzymes are usually synthesized as zymogens and proteolytic cleavage is needed to activate the enzyme. Chymotrypsin is secreted from the pancreas as chymotrypsinogen and it is activated in the digestive tract by the enzyme trypsin. Trypsin cleaves a small peptide from the N-terminal region and converts the inactive chymotrypsinogen to chymotrypsin. (3) Covalent Modification Protein phosphorylation is a major mechanism employed by hormones for intracellular signaling and for regulation of enzymatic activity. The transfer of a phosphate group from ATP to a specific serine or tyrosine residue on the target protein is mediated by protein kinases. Phosphorylation leads to a conformational change and can either activate or inactive the enzyme. Glycogen phosphorylase, which catalyzes the degradation of glycogen, is regulated by phosphorylation in response to the presence of hormones such as insulin and glucagon. (4) Regulatory Proteins Regulatory proteins can modulate the activity of an enzyme. These can either activate or inhibit enzyme activity. When the regulatory protein binds to its target enzyme, a conformation change occurs in the enzyme and this affects the function of the enzyme. Calmodulin, a 17 kD protein serves as a calcium sensor in eukaryotic cells. Ca++ can bind to multiple sites on calmodulin. The Ca++-calmodulin complex in turn binds to many target enzymes and modulate their activity. (5) Allosteric Control The enzyme that catalyzes the first step of a biosynthetic pathway is usually regulated by the ultimate product (Fig. 3.4). This form of regulation

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Enzymology 79

is termed feedback regulation. Feedback regulation allows for quick adjustment to the cells requirement for a particular product and prevents excessive synthesis. Feedback regulation makes use of the properties of allosteric enzymes. Allosteric effectors (allosteric activators and inhibitors) affect the activity of allosteric enzymes. These effectors are molecules other than the substrates and bind at sites separate from the active site of the enzyme. The site where the allosteric effector binds is called the allosteric site (Fig. 3.5). Allosteric enzymes contain multiple subunits. They also exhibit positive cooperativity. Binding of the substrate to one subunit facilitates the binding of substrate to one or more of the remaining subunits. An allosteric activator serves either to activate the enzyme or to stabilize the active state of the enzyme, thus facilitating substrate binding in their own and other

A+B

Enzyme 1

D
(-)

E
Fig. 3.4. Regulation of a pathway by feedback inhibition. C, D and E are intermediates in this hypothetical pathway. The final product, F exerts an inhibitory effect on enzyme 1 which catalyzes the first reaction in the pathway.

s
Enzyme

s s
A

Fig. 3.5. Binding of substrate (S) molecules and effector (A) molecules to an allosteric enzyme. The allosteric site is distinct from the substrate-binding site.

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subunits. Conversely, an allosteric inhibitor will inactive the enzyme and prevent substrate binding. 3.5. The Michaelis-Menten Equation In 1913, Leonor Michaelis and Maud Menten proposed a general theory of enzyme action consistent with observed enzyme kinetics. The enzyme, E associates with its substrate S to form an ES complex with the rate constant of k1. This association is reversible. The ES complex has two fates. It can dissociate to E and S with the rate constant of k2 or the reaction can proceed and the product P is formed (with the rate constant of k3). E+S The Michaelis-Menten equation, v = Vmax [S]/Km + [S] relates the initial velocity (v) of an enzyme reaction to the concentration of the substrate, [S]. Km is defined as the concentration of S at which the velocity of the reaction is at half the maximal. Km is also equal to (k2 + k3)/k1. Vmax is the maximal velocity. A plot of v versus [S] would thus yield a rectangular hyperbola (Fig. 3.6). At infinite substrate concentration, the reaction proceeds under saturation conditions and the reaction velocity approaches Vmax. Rearrangement of the Michaelis-Menten equation can transform it into a linear equation. One such transformation is the Lineweaver-Burk transformation. By taking the reciprocal on both sides, the MichaelisMenten equation is transformed into 1/v = (Km /Vmax) (1/[S]) + 1/Vmax A plot of 1/v versus 1/[S] would yield a straight line and the constants Vmax and Km can be easily determined (Fig. 3.7). The value of Km is dependent on the assay conditions (pH, temperature, presence of inhibitors or activators) but independent of the amount and purity of the enzyme. Km is the substrate concentration at which half the ES E+P

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Enzymology 81

v
Vmax

Vmax/2

Km

[S]

Fig. 3.6. The Michaelis-Menten plot of initial velocity (v) versus substrate concentration, [S]. Vmax is the maximal velocity while Km is the concentration of S at Vmax/2.

1/v
Gradient = Km/Vmax 1/Vmax

1/[S]
-1/Km

Fig. 3.7. The Lineweaver-Burk plot of 1/v versus 1/[S]. v is the initial velocity while [S] is the substrate concentration.

active sites of the enzyme are filled (i.e. Vmax /2). When k2 > k3, then Km > provides a reasonable measure of the affinity of the enzyme for its substrate. A low Km value would indicate that the enzyme has high affinity for the substrate. Km values thus provide a means to compare the affinities of enzymes for their respective substrates.

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The catalytic constant kcat (also known as turnover number) of an enzyme is a measure of its maximal catalytic activity. It is defined as kcat = Vmax/[E]T where [E]T is the total enzyme concentration. kcat equals to k3. It is the number of substrate molecules that is converted into the product by an enzyme molecule in a unit time when the enzyme is fully saturated with its substrate. However under physiological conditions, the substrate concentration is seldom at saturation and kcat values cannot be interpreted meaningfully. The constant kcat/Km is used instead to provide a measure of the enzymes catalytic efficiency under non-saturating substrate concentration ([S] < Km). The above kinetic analysis is for a single substrate reaction. Can it be applied to a multi-substrate reaction? It is beyond the scope of this book to describe such reactions but it suffices to say that Km and Vmax values can still be determined. The usual practice is to determine the velocity of reaction by varying just one substrate while keeping the concentration of all the other cosubstrates constant. In such situations, Michaelis-Menten kinetics will be observed. Are there situations where a deviation from Michaelis-Menten kinetics is observed? A deviation from Michaelis-Menten behaviour is indicative
v
+ AA No AA or AI +AI

[S]

Fig. 3.8. Kinetic profile of an allosteric enzyme in the presence of an allosteric activator (AA) or an allosteric inhibitor (AI). In the absence of an activator or inhibitor, a sigmoidal curve is obtained.

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Enzymology 83

of allosteric regulation (see Sec. 3.4). Hexokinase catalyzes the transfer of a phosphate from ATP to glucose. Hexokinase exists as a number of different tissue-specific isoenzymes. Isoenzymes are proteins with different amino acid sequences, which catalyze the same reaction. They also exhibit different kinetics and regulatory properties. The hexokinases found in most human tissues follow Michaelis-Menten kinetics and a plot of v versus [S] yield a rectangular hyperbola (Fig. 3.6). However, glucokinase (which is the hexokinase isoenzyme found in the liver) behaves differently and a sigmoidal curve is observed. Such sigmoidal curves are characteristic of enzymes that are allosterically regulated (Fig. 3.8). 3.6. Inhibition of Enzyme Activity The activity of an enzyme can be regulated. The various modes of regulation have been described in Sec. 3.4. Besides these regulatory mechanisms, exogenous compounds can also affect the rate of an enzymatic activity. A compound that binds to the enzyme and decreases the velocity of the enzymatic reaction is an inhibitor. Irreversible inhibitors are compounds that form covalent bonds or other strong interactions with the enzyme. Examples of irreversible inhibitors with therapeutic uses include the antibiotic penicillin. Penicillin reacts covalently with bacterial glycopeptide transpeptidase to form the penicilloyl-enzyme complex; rendering the enzyme inactive. Glycopeptide transpeptidase is essential for cell wall biosynthesis. Thus, treatment with penicillin disrupts bacterial cell wall synthesis. Reversible inhibitors, on the other hand, do not form covalent bonds with the enzyme and the inhibitor-enzyme complex can dissociate. There are three forms of reversible inhibition: competitive, uncompetitive and mix (or non-competitive) inhibition. All three types of reversible inhibition can easily be distinguished using the Lineweaver-Burk plot of 1/v versus 1/[S] (Fig. 3.9). (1) Competitive Inhibition A competitive inhibitor is a structural analog of the substrate. It resembles the structure of the substrate. It will thus compete with the substrate for binding to the active site of the enzyme (Fig. 3.10). Competitive inhibition

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can be overcome by increasing the substrate concentration. At high substrate concentration, all the active sites will be occupied by the substrate and the inhibitor will not be able to bind. Hence, competitive inhibition will lead to an increase in the Km value but the maximal velocity (Vmax) remains unchanged (Fig. 3.9). The mode of action of many therapeutic drugs is based on competitive inhibition. The therapeutic drug methotrexate is a structural analog of dihydrofolate. It competes with dihydrofolate for binding to the active site of the enzyme dihydrofolate reductase.
NI

1/v

CI UI No inhibitor

1/Vmax

1/[S]
-1/Km
Fig. 3.9. Lineweaver-Burk plots in the presence of a competitive inhibitor (CI), a noncompetitive inhibitor (NI) and an uncompetitive inhibitor (UI).

+ E

ES

+ EI

Fig. 3.10. Competitive inhibition. The inhibitor (I) competes with the substrate (S) for the active site of the enzyme (E).

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Enzymology 85

+ E

ES

+ ESI

Fig. 3.11. Uncompetitive inhibition. The inhibitor (I) can only bind to the enzyme-substrate (ES) complex.

+ E

ES

+ EI

ESI

Fig. 3.12. Noncompetitive (mixed) inhibition. The inhibitor can bind to either the enzyme (E) or the enzyme-substrate (ES) complex.

(2) Uncompetitive Inhibition An uncompetitive inhibitor behaves differently from a competitive inhibitor. It binds to the enzyme-substrate complex. Binding of the substrate to the enzyme will lead to a conformational change in the enzyme and allow the inhibitor to interact with the enzyme (Fig. 3.11). Formation of the substrate-enzyme-inhibitor complex prevents the reaction from proceeding. Uncompetitive inhibition changes both the Km and Vmax values (Fig. 3.9).

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(3) Mixed Inhibition Mixed inhibition is also known as noncompetitive inhibition. Noncompetitive inhibitors do not compete with the substrate for the active site. Noncompetitive inhibitors bind to the enzyme at a site that is distinct from the active site. Such inhibitors can bind either to the enzyme or the enzyme-substrate complex (Fig. 3.12). Mixed inhibition may or may not alter the Km value of a reaction but a decrease in the Vmax will be observed (Fig. 3.9). 3.7. Industrial Applications of Enzymes Enzymes are useful catalysts. The advantages of enzyme compared to other catalyst include specificity (lack of side reactions) and catalysis under mild conditions (low temperature and atmospheric pressure). The catalytic ability of enzymes has been harnessed and used for centuries. The earliest uses include the use of enzymes present in microorganisms for brewing, bread making and cheese making. These had been carried out before there was any understanding of the biochemical reactions and the enzymes involved. Today, enzymes are used in the chemical, pharmaceutical and food industry. The sales of enzymes for industrial uses have doubled between the years 1983 and 1995. A further doubling is predicted by the year 2005. It is beyond the scope of this section to provide a detailed account of the many and varied uses of enzymes. A selection of examples is used to allow for the appreciation of the industrial and clinical application of enzymes. (1) Brewing An examination of the brewing process will illustrate how enzymes are utilized for the production of beer. Note that for brewing, intact yeast and barley seeds are used and isolation of the required enzymes is not necessary. The starting material is starch, which is present in the barley seed. Starch is a polysaccharide and yeast is unable to ferment starch. This can be overcome by allowing the barley seeds to germinate. During germination, endo-1,3--D-glucanase, amylases and peptidases are released. Starch granules are found within the cells of the endosperm.

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The walls of the endosperm are surrounded by polymers including 1,3-D-glucans. The enzyme endo-1,3--D-glucanase will degrade the glucans so that the amylases can come into contact with the starch granules. Amylases catalyze the hydrolysis of starch to simple sugars including glucose, maltose, and other oligosaccharides. In addition, peptidases are also released and these enzymes will catalyze the degradation of endogenous proteins within the seeds. The resulting peptides and amino acids are utilized by the yeast for growth. The primary role of yeast in brewing is to provide the necessary enzymes for anaerobic glycolysis. Ethanol is the by-product of anaerobic glycolysis. Hence, for brewing, both the germinating barley seed and the yeast provide the full complement of enzymes for converting the starting raw material starch to the final product ethanol. (2) Antibiotic and Therapeutic Products Microorganisms are also utilized for the production of many useful therapeutic products. These include the production of antibiotics such as erythromycin, streptomycin and penicillin by Streptomyces spp and Penicillium spp and the production of lovastatin by Aspergillus terreus. (3) Clinical Applications In clinical applications, enzymes can be used in one of the following manner: as diagnostic tools, as analytical reagents or as therapeutic agents. Currently, more than 20 different enzymes are assayed routinely in clinical chemistry laboratories. Detection of abnormal levels of enzymes in body fluids can aid diagnosis of diseases. Elevated serum levels of aspartate amino transferase (AST), alanine amino transferease (ALT), alkaline phosphatase (ALP) and -glutamyltransferase (GGT) are observed in liver disease. How are these assayed? The key feature when assaying for enzyme activity is the need for an end product that can be easily detected and measured. The product usually absorbs light in the UV or visible range. ALP is assayed by measuring the hydrolysis of 4-nitrophenyl phosphate. The products of this reaction are phosphate and 4-nitrophenol. The latter absorbs at 405 nm. However not all reactions produce at least a product which can be easily measured. An example is the assay for ALT activity.

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Reaction 1: Reaction 2:

alanine + 2-oxoglutarate pyruvate + NADH + H+

pyruvate + glutamate lactate + NAD+

The products pyruvate and glutamate do not absorb light. Hence it is necessary to couple the ALT reaction (Reaction 1) to a second reaction (Reaction 2). The second reaction is catalyzed by lactate dehydrogenase (LDH). Pyruvate, the product from the ALT reaction, is used as a substrate for the subsequent LDH reaction. In the presence of the coenzyme NADH, LDH converts pyruvate to lactate. NADH absorbs at 340 nm while NAD does not. The change in A340 due to oxidation of NADH can be easily measured. Enzymes are also used as analytical reagents in clinical chemistry applications. In the above example, the enzyme LDH is used as an analytical reagent for the assay for ALT. Another example is the use of the enzyme glucose oxidase to assay blood glucose levels. gluconic acid + H2O2 Reaction 1: glucose + H2O + O2 Reaction 2: H2O2 + reduced acceptor H2O + oxidized acceptor Glucose oxidase oxidizes glucose to gluconic acid and hydrogen peroxide (Reaction 1). The hydrogen peroxide produced then oxidizes an acceptor (Reaction 2). The acceptor commonly used is o-dianisidine. Oxidized o-dianisidine absorbs between 425 nm and 475 nm. Enzyme-linked immunosorbant assay (ELISA) also utilizes enzymatic reactions for detection of antigen-antibody interaction (Fig. 3.13). The binding of an antibody to an antigen cannot be easily detected. To facilitate detection, a secondary antibody with an enzyme attached to it is used. Enzymes commonly used for ELISA assays include alkaline phosphatase and horse raddish peroxidase. Both enzymes can react with specific substrates to produce products that are easily detected. Enzymes are also used for a variety of therapeutic purposes (Table 3.2). Streptokinase, urokinase and human tissue plasminogen activator (tPA) are effective activators of plasminogen. These are used as thrombolytic agents and are useful in removing blood clots. Intravenous administration of therapeutic enzyme molecules may elicit allergic, anaphylactic and immunological responses. In addition, the enzyme is usually rapidly removed from circulation and degraded. Much work had been carried out

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to overcome the antigenicity and short plasma half-lives of therapeutic proteins. An example is tPA. Human tPA has been cloned and the recombinant protein is used for myocardical infarction. tPA has poor solubility, is rapidly cleared by the liver and hence a large dose is necessary. However, the side effects of a large dose include bleeding and non-clotting. Recombinant DNA and protein engineering technology have been used to create an improved version of this protein. The engineered tPA is a smaller protein with increased solubility, better pharmacokinetics
P
Enzyme

S
Secondary Ab Primary Ab

Ag

Fig. 3.13. Enzyme-linked immunosorbant (ELIZA) assay. The primary antibody (Ab) recognizes the immobilized antigen (Ag) and the secondary Ab will specifically interact with the primary Ab. These interactions can be detected using an appropriate substrate (S) for the enzyme attached to the secondary Ab. The product (P) formed can be easily measured.

Table 3.2. Therapeutic uses of enzymes. Enzyme Collagenase Trypsin Uricase Hyaluronidase Tissue plasminogen activator Streptokinase Urokinase Factor IXa Application Skin ulcers Anti-inflammatory agent Gout Drug administration Thrombolytic agent Thrombolytic agent Thrombolytic agent Hemophilia B

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and improved specificity and affinity at the clot with little effect on peripheral coagulation. (4) Immobilized Enzymes The attachment of an enzyme to a support will enable us to reuse the enzyme without a tedious recovery process. Enzymes may be immobilized on an inert support in a number of ways. These include adsorption, covalent linkages, entrapment within a matrix and encapsulation. Immobilized -galactosidase has been used to hydrolyze lactose to glucose and galactose. Immobilization is achieved by the adsorption of the enzyme onto silica particles. This immobilized enzyme has been used to produce a mixture of glucose and galactose, which is used as a sweetening agent. Immobilized -galactosidase is also used to remove lactose from milk. Lactose intolerance is a common problem in many parts of the world and this has led to a demand for lactose-free milk. Analytical reagents may also be immobilized. Glucose dehydrogenase can be immobilized onto nylon tubing and this can be used and reused for measuring blood glucose levels. As little as 0.18 mg of enzyme can be used for more than 1000 glucose determinations. The ability to immobilize enzymes and other analytical reagents has also led to a wide range of home testing kits for urine and blood samples. The above examples illustrate how enzymes can be utilized for industrial and clinical applications. In some instances, the use of a suitable microorganism with the appropriate complement of enzymes is sufficient (e.g. brewing) but in other instances, purified enzymes are necessary (e.g. clinical applications). In addition, the progress in genetic engineering and recombinant DNA technology will lead to the production of genetically engineered microorganism and recombinant proteins which have improved features such as enhanced catalytic activity, increased solubility or improved thermal stability. References 1. Steitz TA, Shoham M and Bennett WS Jr. Structural dynamics of yeast hexokinase during catalysis. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 293: 43 52, 1981.

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2. Enzyme Nomenclature. Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Academic Press, London, 1992. 3. Naqui A. Where are the asymptotes of Michaelis-Menten? TiBS 1: 6465, 1986. 4. Godfrey T and West S, eds. Industrial Enzymology. Macmillian, London, 1996. 5. Pollock J. Brewing Science. Academic Press, New York, 1987. 6. Buckel P. Recombinant proteins for therapy. TiPS 17: 450456, 1996. Further Readings 1. Copeland RA. Enzymes A Practical Introduction to Structure, Mechanism and Data Analysis. Wiley-VCH, Inc, USA, 1966. 2. Price NC and Stevens L. Fundamentals of Enzymology The Cell and Molecular Biology of Catalytic Proteins, 3rd ed. Oxford University Press, 2000. Relevant World Wide Web Sites About Enzymes http://tw.expasy.org/enzyme/

Questions for Thought

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(a) Determine the Km and Vmax values for the enzyme. (b) In the presence of an inhibitor I, the Km value increases but the Vmax remains unchanged. (i) What is the nature of this inhibition? (ii) How can this inhibition be overcomed? 2. (a) Two different enzymes: A and B catalyze the same reaction. What is the term used to describe such enzymes? (b) Compound X is a competitive inhibitor of Enzyme A. Another compound, Y interacts with the N-terminal domain of Enzyme A. The N-terminal domain of Enzyme B is different from that of Enzyme A. Kinetic analysis shows that Y is a non competitive inhibitor for Enzyme A. How will X and Y affect the Km and Vmax of Enzyme B? 3. A student was given 2 different preparations of enzyme E and told to determine the Km and Vmax values. Enzyme E was isolated from rat liver using the same protocol on two different occasions. He noticed that the Km values were identical but the Vmax values differ. How can he account for his observations? 4. (a) Explain the principle of coupled enzyme assays. (b) You are interested in determining the concentration of ATP in your sample. With the following reagents (i) (ii) (iii) (iv) glucose glucokinase glucose-6-phosphate dehydrogenase NADP+

outline how you would set up an assay to determine the concentration of ATP in your sample. (c) In place of glucose, you are provided with lactose, galactosidase, ATP and reagents (ii), (iii) and (iv). Using these reagents, illustrate how you can determine the activity of galactosidase in your sample.

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