Enzymes: Mechanism of Action - Structures & Functions of Proteins & Enzymes - Harper’s Illustrated Biochemistry, 29th Edition (2012)

Harper’s Illustrated Biochemistry, 29th Edition (2012)

SECTION I. Structures & Functions of Proteins & Enzymes

Chapter 7. Enzymes: Mechanism of Action

Peter J. Kennelly, PhD & Victor W. Rodwell, PhD

OBJECTIVES

After studying this chapter, you should be able to:

Image Illustrate the structural relationships between B vitamins and coenzymes.

Image Outline the four principal mechanisms by which enzymes achieve catalysis.

Image Describe how an “induced fit” facilitates substrate recognition and catalysis.

Image Outline the underlying principles of enzyme-linked immunoassays. Explain how coupling an enzyme to an NAD(P)+-dependent dehydrogenase can simplify assay of its activity.

Image Identify enzymes and proteins whose plasma levels are used for the diagnosis and prognosis of a myocardial infarction.

Image Describe the application of restriction endonucleases and of restriction fragment length polymorphisms in the detection of genetic diseases.

Image Explain the utility of site-directed mutagenesis for the identification of residues involved in catalysis, in the recognition of substrates or allosteric effectors, or in the mechanism of enzyme action.

Image Describe how the addition of fused affinity “tags” via recombinant DNA technology can facilitate purification of a protein expressed from its cloned gene.

Image Indicate the function of specific proteases in the purification of affinity-tagged enzymes.

Image Discuss the events that led to the discovery that RNAs can act as enzymes.

BIOMEDICAL IMPORTANCE

Enzymes are biologic polymers that catalyze the chemical reactions that make life, as we know it, possible. The presence and maintenance of a complete and balanced set of enzymes is essential for the breakdown of nutrients to supply energy and chemical building blocks; the assembly of those building blocks into proteins, DNA, membranes, cells, and tissues; and the harnessing of energy to power cell motility, neural function, and muscle contraction. The vast majority of enzymes are proteins. Notable exceptions include ribosomal RNAs and a handful of self-cleaving or self-splicing RNA molecules known collectively as ribozymes. The ability to assay the activity of specific enzymes in blood, other tissue fluids, or cell extracts aids in the diagnosis and prognosis of disease. Deficiencies in the quantity or catalytic activity of key enzymes can result from genetic defects, nutritional deficits, or toxins.

Defective enzymes can result from genetic mutations or infection by viral or bacterial pathogens (eg, Vibrio cholerae). Medical scientists address imbalances in enzyme activity by using pharmacologic agents to inhibit specific enzymes and are investigating gene therapy as a means to remedy deficits in enzyme level or function.

In addition to serving as the catalysts for all metabolic processes, their impressive catalytic activity, substrate specificity, and stereospecificity enable enzymes to fulfill key roles in other processes related to human health and well-being. The absolute stereospecificity of enzymes is of a particular value for use as soluble or immobilized catalysts for specific reactions in the synthesis of a drug or antibiotic. Proteases and amylases augment the capacity of detergents to remove dirt and stains. Enzymes play an important role in producing or enhancing the nutrient value of food products for both humans and animals. The protease rennin, for example, is utilized in the production of cheeses while lactase is employed to remove lactose from milk for the benefit of lactose-intolerant persons deficient in this hydrolytic enzyme (Chapter 43).

ENZYMES ARE EFFECTIVE & HIGHLY SPECIFIC CATALYSTS

The enzymes that catalyze the conversion of one or more compounds (substrates) into one or more different compounds (products) enhance the rates of the corresponding non-catalyzed reaction by factors of at least 106. Like all catalysts, enzymes are neither consumed nor permanently altered as a consequence of their participation in a reaction.

In addition to being highly efficient, enzymes are also extremely selective catalysts. Unlike most catalysts used in synthetic chemistry, enzymes are specific both for the type of reaction catalyzed and for a single substrate or a small set of closely related substrates. Enzymes are also stereospecific catalysts and typically catalyze reactions of only one stereoisomer of a given compound—for example, D- but not L-sugars, L- but not D-amino acids. Since they bind substrates through at least “three points of attachment,” enzymes can even convert non-chiral substrates to chiral products. Figure 7–1 illustrates why the enzyme-catalyzed reduction of the nonchiral substrate pyruvate produces only L-lactate, not a racemic mixture of D- and L-lactate. The exquisite specificity of enzyme catalysts imbues living cells with the ability to simultaneously conduct and independently control a broad spectrum of chemical processes.

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FIGURE 7–1 Planar representation of the “three-point attachment” of a substrate to the active site of an enzyme. Although atoms 1 and 4 are identical, once atoms 2 and 3 are bound to their complementary sites on the enzyme, only atom 1 can bind. Once bound to an enzyme, apparently identical atoms thus may be distinguishable, permitting a stereospecific chemical change.

ENZYMES ARE CLASSIFIED BY REACTION TYPE

The commonly used names for most enzymes describe the type of reaction catalyzed, followed by the suffix -ase. For example, dehydrogenases remove hydrogen atoms, proteases hydrolyze proteins, and isomerases catalyze rearrangements in configuration. Modifiers may precede the name to indicate the substrate (xanthine oxidase), the source of the enzyme (pancreatic ribonuclease), its regulation (hormone-sensitive lipase), or a feature of its mechanism of action (cysteine protease). Where needed, alphanumeric designators are added to identify multiple forms of an enzyme (eg, RNA polymerase III; protein kinase Cβ).

To address ambiguities, the International Union of Biochemists (IUB) developed an unambiguous system of enzyme nomenclature in which each enzyme has a unique name and code number that identify the type of reaction catalyzed and the substrates involved. Enzymes are grouped into the following six classes.

1. Oxidoreductases—enzymes that catalyze oxidations and reductions.

2. Transferases— enzymes that catalyze transfer of moieties such as glycosyl, methyl, or phosphoryl groups.

3. Hydrolases— enzymes that catalyze hydrolytic cleavage of C—C, C—O, C—N and other covalent bonds.

4. Lyases— enzymes that catalyze cleavage of C—C, C—O, C—N and other covalent bonds by atom elimination, generating double bonds.

5. Isomerases—enzymes that catalyze geometric or structural changes within a molecule.

6. Ligases—enzymes that catalyze the joining together (ligation) of two molecules in reactions coupled to the hydrolysis of ATP.

Despite the clarity of the IUB system, the names are lengthy and relatively cumbersome, so we generally continue to refer to enzymes by their traditional, albeit sometimes ambiguous names. The IUB name for hexokinase illustrates both the clarity of the IUB system and its complexities. The IUB name of hexokinase is ATP:D-hexose 6-phosphotransferase E.C. 2.7.1.1. This name identifies hexokinase as a member of class 2 (transferases), subclass 7 (transfer of a phosphoryl group), subsubclass 1 (alcohol is the phosphoryl acceptor), and “hexose-6” indicates that the alcohol phosphorylated is on carbon six of a hexose. However, we continue to call it hexokinase.

PROSTHETIC GROUPS, COFACTORS, & COENZYMES PLAY IMPORTANT ROLES IN CATALYSIS

Many enzymes contain small nonprotein molecules and metal ions that participate directly in substrate binding or in catalysis. Termed prosthetic groups, cofactors, and coenzymes, these extend the repertoire of catalytic capabilities beyond those afforded by the limited number of functional groups present on the aminoacyl side chains of peptides.

Prosthetic Groups Are Tightly Integrated Into an Enzyme’s Structure

Prosthetic groups are tightly and stably incorporated into a protein’s structure by covalent or noncovalent forces. Examples include pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamin pyrophosphate, biotin, and the metal ions of Co, Cu, Mg, Mn, and Zn. Metals are the most common prosthetic groups. The roughly one-third of all enzymes that contain tightly bound metal ions are termed metalloenzymes. Metal ions that participate in redox reactions generally are complexed to prosthetic groups such as heme (Chapter 6) or iron–sulfur clusters (Chapter 12). Metals also may facilitate the binding and orientation of substrates, the formation of covalent bonds with reaction intermediates (Co2+ in coenzyme B12), or by acting as Lewis acids or bases to render substrates more electrophilic (electron-poor) or nucleophilic (electron-rich), and hence more reactive.

Cofactors Associate Reversibly With Enzymes or Substrates

Cofactors serve functions similar to those of prosthetic groups, but bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP. Unlike the stably associated prosthetic groups, cofactors must be present in the medium surrounding the enzyme for catalysis to occur. The most common cofactors also are metal ions. Enzymes that require a metal ion cofactor are termed metal-activated enzymes to distinguish them from the metalloenzymes for which metal ions serve as prosthetic groups.

Coenzymes Serve as Substrate Shuttles

Coenzymes serve as recyclable shuttles—or group transfer agents—that transport many substrates from one point within the cell to another. The function of these shuttles is twofold. First, they stabilize species such as hydrogen atoms (FADH) or hydride ions (NADH) that are too reactive to persist for any significant time period in the presence of the water or organic molecules that permeate the cell interior. They also serve as an adaptor or handle that facilitates the recognition and binding of small chemical groups, such as acetate (coenzyme A), by their target enzymes. Other chemical moieties transported by coenzymes include methyl groups (folates) and oligosaccharides (dolichol).

Many Coenzymes, Cofactors & Prosthetic Groups Are Derivatives of B Vitamins

The water-soluble B vitamins supply important components of numerous coenzymes. Several coenzymes contain, in addition, the adenine, ribose, and phosphoryl moieties of AMP or ADP (Figure 7–2). Nicotinamide is a component of the redox coenzymes NAD and NADP, whereas riboflavin is a component of the redox coenzymes FMN and FAD. Pantothenic acid is a component of the acyl group carrier coenzyme A. As its pyrophosphate, thiamin participates in decarboxylation of α-keto acids, and the folic acid and cobamide coenzymes function in one-carbon metabolism.

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FIGURE 7–2 Structure of NAD+ and NADP+. For NAD+, Image. For NADP+, Image

CATALYSIS OCCURS AT THE ACTIVE SITE

An important early 20th-century insight into enzymic catalysis sprang from the observation that the presence of substrates renders enzymes more resistant to the denaturing effects of elevated temperatures. This observation led Emil Fischer to propose that enzymes and their substrates interact to form an enzyme–substrate (ES) complex whose thermal stability was greater than that of the enzyme itself. This insight profoundly shaped our understanding of both the chemical nature and kinetic behavior (Chapter 8) of enzymic catalysis.

Fischer reasoned that the exquisitely high specificity with which enzymes discriminate their substrates when forming an ES complex was analogous to the manner in which a mechanical lock distinguishes the proper key. In most enzymes, the “lock” is formed by a cleft or pocket on the protein’s surface that forms part of a region called the active site (Figures 5–6 and 5–8). As implied by the adjective “active,” the active site is much more than simply a recognition site for binding substrates. Within the active site, substrates are brought into close proximity to one another in optimal alignment with the cofactors, prosthetic groups, and amino acid side chains responsible for catalyzing their chemical transformation into products (Figure 7–3). Catalysis is further enhanced by the capacity of the active site to shield substrates from water and generate an environment whose polarity, hydrophobicity, acidity, or alkalinity can differ markedly from that of the surrounding cytoplasm.

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FIGURE 7–3 Two-dimensional representation of a dipeptide substrate, glycyl-tyrosine, bound within the active site of carboxypeptidase A.

ENZYMES EMPLOY MULTIPLE MECHANISMS TO FACILITATE CATALYSIS

Enzymes use various combinations of four general mechanisms to achieve dramatic catalytic enhancement of the rates of chemical reactions.

Catalysis by Proximity

For molecules to react, they must come within bond-forming distance of one another. The higher their concentration, the more frequently they will encounter one another, and the greater will be the rate of their reaction. When an enzyme binds substrate molecules at its active site, it creates a region of high local substrate concentration. This environment also orients the substrate molecules spatially in a position ideal for them to interact, resulting in rate enhancements of at least a thousandfold.

Acid–Base Catalysis

The ionizable functional groups of aminoacyl side chains and (where present) of prosthetic groups contribute to catalysis by acting as acids or bases. Acid-base catalysis can be either specific or general. By “specific” we mean only protons (H3O+) or OH ions. In specific acid catalysis or specific base catalysis, the rate of reaction is sensitive to changes in the concentration of protons of but independent of the concentrations of other acids (proton donors) or bases (proton acceptors) present in the solution or at the active site. Reactions whose rates are responsive to all the acids or bases present are said to be subject to general acid catalysis or general base catalysis.

Catalysis by Strain

Enzymes that catalyze lytic reactions that involve breaking a covalent bond typically bind their substrates in a conformation that is somewhat unfavorable for the bond that will undergo cleavage. This conformation mimics that of the transition state intermediate, a transient species that represents the transition state, or half-way point, in the transformation of substrates to products. The resulting strain stretches or distorts the targeted bond, weakening it and making it more vulnerable to cleavage. Nobel Laureate Linus Pauling was the first to suggest a role for transition state stabilization as a general mechanism by which enzymes accelerate the rates of chemical reactions. Knowledge of the transition state of an enzyme-catalyzed reaction is frequently exploited by chemists to design and create more effective enzyme inhibitors, called transition state analogs, as potential pharmacophores.

Covalent Catalysis

The process of covalent catalysis involves the formation of a covalent bond between the enzyme and one or more substrates. The modified enzyme then becomes a reactant. Covalent catalysis introduces a new reaction pathway whose activation energy is lower—and the reaction therefore is faster—than the reaction pathway in homogeneous solution. Chemical modification of the enzyme is, however, transient. Completion of the reaction returns the enzyme to its original unmodified state. Its role thus remains catalytic. Covalent catalysis is particularly common among enzymes that catalyze group transfer reactions. Residues on the enzyme that participate in covalent catalysis generally are cysteine or serine and occasionally histidine. Covalent catalysis often follows a “ping-pong” mechanism—one in which the first substrate is bound and its product released prior to the binding of the second substrate (Figure 7–4).

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FIGURE 7–4 “Ping-pong” mechanism for transamination. E—CHO and E—CH2NH2 represent the enzyme-pyridoxal phosphate and enzyme-pyridoxamine complexes, respectively. (Ala, alanine; Glu, glutamate; KG, α-ketoglutarate; Pyr, pyruvate.)

SUBSTRATES INDUCE CONFORMATIONAL CHANGES IN ENZYMES

While Fischer’s “lock and key model” accounted for the exquisite specificity of enzyme-substrate interactions, the implied rigidity of the enzyme’s active site failed to account for the dynamic changes that we now know accompany catalysis. This drawback was addressed by Daniel Koshland’s induced fit model, which states that when substrates approach and bind to an enzyme they induce a conformational change analogous to placing a hand (substrate) into a glove (enzyme) (Figure 7–5). The enzyme in turn induces reciprocal changes in its substrates, harnessing the energy of binding to facilitate the transformation of substrates into products. The induced fit model has been amply confirmed by biophysical studies of enzyme motion during substrate binding.

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FIGURE 7–5 Two-dimensional representation of Koshland’s induced fit model of the active site of a lyase. Binding of the substrate A—B induces conformational changes in the enzyme that align catalytic residues which participate in catalysis and strains the bond between A and B, facilitating its cleavage.

HIV PROTEASE ILLUSTRATES ACID–BASE CATALYSIS

Enzymes of the aspartic protease family, which includes the digestive enzyme pepsin, the lysosomal cathepsins, and the protease produced by the human immunodeficiency virus (HIV) share a common catalytic mechanism. Catalysis involves two conserved aspartyl residues, which act as acid–base catalysts. In the first stage of the reaction, an aspartate functioning as a general base (Asp X, Figure 7–6) extracts a proton from a water molecule, making it more nucleophilic. The resulting nucleophile then attacks the electrophilic carbonyl carbon of the peptide bond targeted for hydrolysis, forming a tetrahedral transition state intermediate. A second aspartate (Asp Y, Figure 7–6) then facilitates the decomposition of this tetrahedral intermediate by donating a proton to the amino group produced by rupture of the peptide bond. The two different active site aspartates can act simultaneously as a general base or as a general acid because their immediate environment favors ionization of one, but not the other.

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FIGURE 7–6 Mechanism for catalysis by an aspartic protease such as HIV protease. Curved arrows indicate directions of electron movement. Image Aspartate X acts as a base to activate a water molecule by abstracting a proton. Image The activated water molecule attacks the peptide bond, forming a transient tetrahedral intermediate. Image Aspartate Y acts as an acid to facilitate breakdown of the tetrahedral intermediate and release of the split products by donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its initial state.

CHYMOTRYPSIN & FRUCTOSE-2, 6-BISPHOSPHATASE ILLUSTRATE COVALENT CATALYSIS

Chymotrypsin

While catalysis by aspartic proteases involves the direct hydrolytic attack of water on a peptide bond, catalysis by the serine protease chymotrypsin involves prior formation of a covalent acyl-enzyme intermediate. A highly reactive seryl residue, serine 195, participates in a charge-relay network with histidine 57 and aspartate 102. While these three residues are far apart in primary structure, in the active site of the mature, folded protein they are within bond-forming distance of one another. Aligned in the order Asp 102-His 57-Ser 195, they constitute a “charge-relay network” that functions as a “proton shuttle.”

Binding of substrate initiates proton shifts that in effect transfer the hydroxyl proton of Ser 195 to Asp 102 (Figure 7–7). The enhanced nucleophilicity of the seryl oxygen facilitates its attack on the carbonyl carbon of the peptide bond of the substrate, forming a covalent acyl-enzyme intermediate. The proton on Asp 102 then shuttles via His 57 to the amino group liberated when the peptide bond is cleaved. The portion of the original peptide with a free amino group then leaves the active site and is replaced by a water molecule. The charge-relay network now activates the water molecule by withdrawing a proton through His 57 to Asp 102. The resulting hydroxide ion attacks the acyl-enzyme intermediate and a reverse proton shuttle returns a proton to Ser 195, restoring its original state. While modified during the process of catalysis, chymotrypsin emerges unchanged on completion of the reaction. The proteases trypsin and elastase employ a similar catalytic mechanism, but the numbers of the residues in their Ser-His-Asp proton shuttles differ.

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FIGURE 7–7 Catalysis by chymotrypsin. Image The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. Image Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. Image Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. Image His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. Image The charge-relay system donates a proton to Ser 195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptideImage.

Fructose-2,6-Bisphosphatase

Fructose-2,6-bisphosphatase, a regulatory enzyme of gluconeogenesis (Chapter 20), catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose 2,6-bisphosphate. Figure 7–8 illustrates the roles of seven active site residues. Catalysis involves a “catalytic triad” of one Glu and two His residues and a covalent phosphohistidyl intermediate.

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FIGURE 7–8 Catalysis by fructose-2,6-bisphosphatase. (1) Lys 356 and Arg 257, 307, and 352 stabilize the quadruple negative charge of the substrate by charge-charge interactions. Glu 327 stabilizes the positive charge on His 392. (2) The nucleophile His 392 attacks the C-2 phosphoryl group and transfers it to His 258, forming a phosphoryl-enzyme intermediate. Fructose 6-phosphate now leaves the enzyme. (3) Nucleophilic attack by a water molecule, possibly assisted by Glu 327 acting as a base, forms inorganic phosphate. (4) Inorganic orthophosphate is released from Arg 257 and Arg 307. (Reproduced, with permission, from Pilkis SJ, et al: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: A metabolic signaling enzyme. Annu Rev Biochem 1995;64:799. © 1995 by Annual Reviews, www.annualreviews.org.)

CATALYTIC RESIDUES ARE HIGHLY CONSERVED

Members of an enzyme family such as the aspartic or serine proteases employ a similar mechanism to catalyze a common reaction type, but act on different substrates. Most enzyme families arose through gene duplication events that created a second copy of the gene that encodes a particular enzyme. The proteins encoded by the two genes can then evolve independently to recognize different substrates—resulting, for example, in chymotrypsin, which cleaves peptide bonds on the carboxyl terminal side of large hydrophobic amino acids, and trypsin, which cleaves peptide bonds on the carboxyl terminal side of basic amino acids. Proteins that diverged from a common ancestor are said to be homologous to one another. The common ancestry of enzymes can be inferred from the presence of specific amino acids in the same position in each family member. These residues are said to be conserved residues. Table 7–1 illustrates the primary structural conservation of two components of the charge-relay network for several serine proteases. Among the most highly conserved residues are those that participate directly in catalysis.

TABLE 7–1 Amino Acid Sequences in the Neighborhood of the Catalytic Sites of Several Bovine Proteases

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ISOZYMES ARE DISTINCT ENZYME FORMS THAT CATALYZE THE SAME REACTION

Higher organisms often elaborate several physically distinct versions of a given enzyme, each of which catalyzes the same reaction. Like the members of other protein families, these protein catalysts or isozymes arise through gene duplication. Isozymes may exhibit subtle differences in properties such as sensitivity to particular regulatory factors (Chapter 9) or substrate affinity (eg, hexokinase and glucokinase) that adapt them to specific tissues or circumstances. Some isozymes may also enhance survival by providing a “backup” copy of an essential enzyme.

THE CATALYTIC ACTIVITY OF ENZYMES FACILITATES THEIR DETECTION

The relatively small quantities of enzymes present in cells complicate determination of their presence and concentration. However, the amplification conferred by their ability to rapidly transform thousands of molecules of a specific substrate into products imbues each enzyme with the ability to reveal its presence. Assays of the catalytic activity of enzymes are frequently used in research and clinical laboratories. Under appropriate conditions (see Chapter 8), the rate of the catalytic reaction being monitored is proportionate to the amount of enzyme present, which allows its concentration to be inferred.

Single-Molecule Enzymology

The limited sensitivity of traditional enzyme assays necessitates the use of a large group, or ensemble, of enzyme molecules in order to produce measurable quantities of product. The data obtained thus reflect the average catalytic capability of individual molecules. Recent advances in nanotechnology have made it possible to observe, usually by fluorescence microscopy, catalytic events involving individual enzyme and substrate molecules. Consequently, scientists can now measure the rate of single catalytic events and sometimes the individual steps in catalysis by a process called single-molecule enzymology (Figure 7–9).

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FIGURE 7–9 Direct observation of single DNA cleavage events catalyzed by a restriction endonuclease. DNA molecules immobilized to beads (pale yellow) are placed in a flowing stream of buffer (black arrows), which causes them to assume an extended conformation. Cleavage at one of the restriction sites (orange) by an endonuclease leads to a shortening of the DNA molecule, which can be observed directly in a microscope since the nucleotide bases in DNA are fluorescent. Although the endonuclease (red) does not fluoresce, and hence is invisible, the progressive manner in which the DNA molecule is shortened (1 → 4) reveals that the endonuclease binds to the free end of the DNA molecule and moves along it from site to site.

Drug Discovery Requires Enzyme Assays Suitable for High-Throughput Screening

Enzymes constitute one of the primary classes of biomolecules targeted for the development of drugs and other therapeutic agents. Many antibiotics, for example, inhibit enzymes that are unique to microbial pathogens. The discovery of new drugs is greatly facilitated when a large number of potential phar-macophores can be assayed in a rapid, automated fashion—a process referred to as high-throughput screening. High-throughput screening (HTS) takes advantage of recent advances in robotics, optics, data processing, and microfluidics to conduct and analyze many thousands of simultaneous assays of the activity of a given enzyme. The most commonly used high-throughput screening devices employ 4–100 μL volumes in 96, 384, or 1536 well plastic plates and fully automated equipment capable of dispensing substrates, coenzymes, enzymes, and potential inhibitors in a multiplicity of combinations and concentrations. High-throughput screening is ideal for surveying the numerous products of combinatorial chemistry, the simultaneous synthesis of large libraries of chemical compounds that contain all possible combinations of a set of chemical precursors. Enzyme assays that produce a chromagenic or fluorescent product are ideal, since optical detectors are readily engineered to permit the rapid analysis of multiple samples. At present, the sophisticated equipment required for truly large numbers of assays is available only in pharmaceutical houses, government-sponsored laboratories, and research universities. As described in Chapter 8, its principal use is the analysis of inhibitory compounds with ultimate potential for use as drugs.

Enzyme-Linked Immunoassays

The sensitivity of enzyme assays can be exploited to detect proteins that lack catalytic activity. Enzyme-linked immuno-sorbent assays (ELISAs) use antibodies covalently linked to a “reporter enzyme” such as alkaline phosphatase or horseradish peroxidase whose products are readily detected, generally by the absorbance of light or by fluorescence. Serum or other biologic samples to be tested are placed in a plastic microtiter plate, where the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then “blocked” by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody is then determined by adding the substrate for the reporter enzyme.

NAD(P)-Dependent Dehydrogenases Are Assayed Spectrophotometrically

The physicochemical properties of the reactants in an enzyme-catalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the ability of a substrate or product to absorb light. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb light at a wavelength of 340 nm, whereas their oxidized forms NAD(P)+ do not (Figure 7–10). When NAD(P)+ is reduced, the absor-bance at 340 nm therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, for a dehydrogenase that catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 nm will be observed. In each case, the rate of change in optical density at 340 nm will be proportionate to the quantity of the enzyme present.

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FIGURE 7–10 Absorption spectra of NAD+ and NADH. Densities are for a 44 mg/L solution in a cell with a 1 cm light path. NADP+ and NADPH have spectra analogous to NAD+ and NADH, respectively.

Many Enzymes Are Assayed by Coupling to a Dehydrogenase

The assay of enzymes whose reactions are not accompanied by a change in absorbance or fluorescence is generally more difficult. In some instances, the product or remaining substrate can be transformed into a more readily detected compound. In other instances, the reaction product may have to be separated from unreacted substrate prior to measurement. An alternative strategy is to devise a synthetic substrate whose product absorbs light or fluoresces. For example, p-nitrophenyl phosphate is an artificial substrate for certain phosphatases and for chymotrypsin that does not absorb visible light. However, following hydrolysis, the resulting p-nitrophenylate anion absorbs light at 419 nm.

Another quite general approach is to employ a “coupled” assay (Figure 7–11). Typically, a dehydrogenase whose substrate is the product of the enzyme of interest is added in catalytic excess. The rate of appearance or disappearance of NAD(P)H then depends on the rate of the enzyme reaction to which the dehydrogenase has been coupled.

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FIGURE 7–11 Coupled enzyme assay for hexokinase activity. The production of glucose 6-phosphate by hexokinase is coupled to the oxidation of this product by glucose-6-phosphate dehydrogenase in the presence of added enzyme and NADP+. When an excess of glucose-6-phosphate dehydrogenase is present, the rate of formation of NADPH, which can be measured at 340 nm, is governed by the rate of formation of glucose-6-phosphate by hexokinase.

THE ANALYSIS OF CERTAIN ENZYMES AIDS DIAGNOSIS

The analysis of enzymes in blood plasma has played a central role in the diagnosis of several disease processes. Many enzymes are functional constituents of blood. Examples include pseudocholinesterase, lipoprotein lipase, and components of the cascade that trigger blood clotting and clot dissolution. Other enzymes are released into plasma following cell death or injury. While these latter enzymes perform no physiologic function in plasma, their appearance or levels can assist in the diagnosis and prognosis of diseases and injuries affecting specific tissues. Following injury, the plasma concentration of a released enzyme may rise early or late, and may decline rapidly or slowly. Proteins from the cytoplasm tend to appear more rapidly than those from subcellular organelles. The speed with which enzymes and other proteins are removed from plasma varies with their susceptibility to proteolysis and permeability through renal glomeruli.

Quantitative analysis of the activity of released enzymes or other proteins, typically in plasma or serum but also in urine or various cells, provides information concerning diagnosis, prognosis, and response to treatment. Assays of enzyme activity typically employ standard kinetic assays of initial reaction rates. Table 7–2 lists several enzymes of value in clinical diagnosis. These enzymes are, however, not absolutely specific for the indicated disease. For example, elevated blood levels of prostatic acid phosphatase are associated typically with prostate cancer, but also with certain other cancers and non-cancerous conditions. Consequently, enzyme assay data must be considered together with other factors elicited through a comprehensive clinical examination. Factors to be considered in interpreting enzyme data include patient age, sex, prior history, possible drug use, and the sensitivity and the diagnostic specificity of the enzyme test.

TABLE 7–2 Principal Serum Enzymes Used in Clinical Diagnosis

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Enzymes Assist Diagnosis of Myocardial Infarction

An enzyme useful for diagnostic enzymology should be relatively specific for the tissue or organ under study, should appear in the plasma or other fluid at a time useful for diagnosis (the “diagnostic window”), and should be amenable to automated assay. The enzymes used to confirm a myocardial infarction (MI) illustrate the concept of a “diagnostic window,” and provide a historical perspective on the use of different enzymes for this purpose.

Detection of an enzyme must be possible within a few hours of an MI to confirm a preliminary diagnosis and permit initiation of appropriate therapy. Enzymes that only appear in the plasma 12 h or more following injury are thus of limited utility. The first enzymes used to diagnose MI were aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase. AST and ALT proved less than ideal, however, as they appear in plasma relatively slowly and are not specific to heart muscle. While LDH also is released relatively slowly into plasma, it offered the advantage of tissue specificity as a consequence of its quaternary structure.

Lactate dehydrogenase (LDH) is a tetrameric enzyme consisting of two monomer types: H (for heart) and M (for muscle) that combine to yield five LDH isozymes: HHHH (I1), HHHM (I2), HHMM (I3), HMMM (I4), and MMMM (I5). Tissue-specific expression of the H and M genes determines the relative proportions of each subunit in different tissues. Isozyme I1 predominates in heart tissue, and isozyme I5 in the liver. Thus, tissue injury releases a characteristic pattern of LDH isozymes that can be separated by electrophoresis and detected using a coupled assay (Figure 7–12). Today, LDH has been superseded as a marker for MI by other proteins that appear more rapidly in plasma.

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FIGURE 7–12 Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human serum. LDH isozymes of serum were separated by electrophoresis and visualized using the coupled reaction scheme shown on the left. (NBT, nitroblue tetrazolium; PMS, phenazine methylsulfate.) At right is shown the stained electropherogram. Pattern A is serum from a patient with a myocardial infarct; B is normal serum; and C is serum from a patient with liver disease. Arabic numerals denote specific LDH isozymes.

Creatine kinase (CK) has three isozymes: CK-MM (skeletal muscle), CK-BB (brain), and CK-MB (heart and skeletal muscle). CK-MB has a useful diagnostic window. It appears within 4–6 h of an MI, peaks at 24 h, and returns to baseline by 48–72 h. As for LDH, individual CK isozymes are separable by electrophoresis, thus facilitating detection. Assay of plasma CK levels continues in use to assess skeletal muscle disorders such as Duchene muscular dystrophy. Today, however, in most clinical laboratories the measurement of plasma troponin levels has replaced CK as the preferred diagnostic marker for MI.

Troponins

Troponin is a complex of three proteins involved in muscle contraction in skeletal and cardiac muscle but not in smooth muscle (see Chapter 49). Immunological measurement of plasma levels of cardiac troponins I and T provide sensitive and specific indicators of damage to heart muscle. Troponin levels rise for 2–6 h after an MI and remain elevated for 4–10 days. In addition to MI, other heart muscle damage also elevates serum troponin levels. Cardiac troponins thus serve as a marker of all heart muscle damage. The search for additional markers for heart disease, such as ischemia-modified albumin, and the simultaneous assessment of a spectrum of diagnostic markers via proteomics, continues to be an active area of clinical research.

Enzymes also can be employed in the clinical laboratory as tools for determining the concentration of critical metabolites. For example, glucose oxidase is frequently utilized to measure plasma glucose concentration. Enzymes are employed with increasing frequency as tools for the treatment of injury and disease. Tissue plasminogen activator (tPA) or streptokinase is used in the treatment of acute MI, while trypsin has been used in the treatment of cystic fibrosis (see Chapter 54).

ENZYMES FACILITATE DIAGNOSIS OF GENETIC AND INFECTIOUS DISEASES

Many diagnostic techniques take advantage of the specificity and efficiency of the enzymes that act on oligonucleotides such as DNA (Chapter 39). Enzymes known as restriction endonucleases, for example, cleave double-stranded DNA at sites specified by a sequence of four, six, or more base pairs called restriction sites. Cleavage of a sample of DNA with a restriction enzyme produces a characteristic set of smaller DNA fragments (see Chapter 39). Deviations in the normal product pattern, called restriction fragment length polymorphisms (RFLPs), occur if a mutation renders a restriction site unrecognizable to its cognate restriction endonuclease or, alternatively, generates a new recognition site. RFLPs are currently utilized to facilitate prenatal detection of a number of hereditary disorders, including sickle cell trait, beta-thalassemia, infant phenylketonuria, and Huntington’s disease.

The polymerase chain reaction (PCR) employs a thermostable DNA polymerase and appropriate oligonucleotide primers to produce thousands of copies of a defined segment of DNA from a minute quantity of starting material (see Chapter 39). PCR enables medical, biological, and forensic scientists to detect and characterize DNA present initially at levels too low for direct detection. In addition to screening for genetic mutations, PCR can be used to detect and identify pathogens and parasites such as Trypanosoma cruzi, the causative agent of Chagas’ disease, and Neisseria meningitides, the causative agent of bacterial meningitis, through the selective amplification of their DNA.

RECOMBINANT DNA PROVIDES AN IMPORTANT TOOL FOR STUDYING ENZYMES

Recombinant DNA technology has emerged as an important asset in the study of enzymes. Highly purified samples of enzymes are necessary for the study of their structure and function. The isolation of an individual enzyme, particularly one present in low concentration, from among the thousands of proteins present in a cell can be extremely difficult. If the gene for the enzyme of interest has been cloned, it generally is possible to produce large quantities of its encoded protein in Escherichia coli or yeast. However, not all animal proteins can be expressed in an active form in microbial cells, nor do microbes perform certain post-translational processing tasks. For these reasons, a gene may be expressed in cultured animal cell systems employing the baculovirus expression vector to transform cultured insect cells. For more details concerning recombinant DNA techniques, see Chapter 39.

Recombinant Fusion Proteins Are Purified by Affinity Chromatography

Recombinant DNA technology can also be used to create modified proteins that are readily purified by affinity chromatography. The gene of interest is linked to an oligonucleotide sequence that encodes a carboxyl or amino terminal extension to the encoded protein. The resulting modified protein, termed as a fusion protein, contains a domain tailored to interact with a specific affinity support. One popular approach is to attach an oligonucleotide that encodes six consecutive histidine residues. The expressed “His tag” protein binds to chromatographic supports that contain an immobilized divalent metal ion such as Ni2+ or Cd2+. Alternatively, the substrate-binding domain of glutathione S-transferase (GST) can serve as a “GST tag.” Figure 7–13 illustrates the purification of a GST-fusion protein using an affinity support containing bound glutathione. Fusion proteins also often encode a cleavage site for a highly specific protease such as thrombin in the region that links the two portions of the protein. This permits removal of the added fusion domain following affinity purification.

Image

FIGURE 7–13 Use of glutathione S-transferase (GST) fusion proteins to purify recombinant proteins. (GSH, glutathione.)

Site-Directed Mutagenesis Provides Mechanistic Insights

Once the ability to express a protein from its cloned gene has been established, it is possible to employ site-directed mutagenesis to change specific aminoacyl residues by altering their codons. Used in combination with kinetic analyses and x-ray crystallography, this approach facilitates identification of the specific roles of given aminoacyl residues in substrate binding and catalysis. For example, the inference that a particular aminoacyl residue functions as a general acid can be tested by replacing it with an aminoacyl residue incapable of donating a proton.

RIBOZYMES: ARTIFACTS FROM THE RNA WORLD

Cech Discovered the First Catalytic RNA Molecule

The participation of enzyme catalysts in the posttranslational maturation of certain proteins has analogies in the RNA world. Many RNA molecules undergo processing that both removes segments of oligonucleotide and re-ligates the remaining segments to form the mature product (Chapter 36). Not all of these catalysts are proteins, however. While examining the processing of ribosomal RNA (rRNA) molecules in the ciliated protozoan Tetrahymena,Thomas Cech and his coworkers observed, in the early 1980s, that processing of the 26S rRNA proceeded smoothly in vitro even in the total absence of protein. The source of this splicing activity was traced to a 413 bp catalytic segment that retained its catalytic activity even when replicated in E. coli (Chapter 39). Prior to that time, polynucleotides had been thought to serve solely as information storage and transmission entities, and that catalysis was restricted solely to proteins.

Several other ribozymes have since been discovered. The vast majority catalyze nucleophilic displacement reactions that target the phosphodiester bonds of the RNA backbone. In small self-cleaving RNAs, such as hammerhead or hepatitis delta virus RNA, the attacking nucleophile is water and the result is hydrolysis. For the large group I intron ribozymes, the attacking nucleophile is the 3′-hydroxyl of the terminal ribose of another segment of RNA and the result is a splicing reaction.

The Ribosome—The Ultimate Ribozyme

The ribozyme was the first recognized “molecular machine.” A massive complex comprised of scores of protein subunits and several large ribosomal RNA molecules, the ribosome performs the vitally important and highly complex process of synthesizing long polypeptide chains following the instructions encoded in messenger RNA molecules (Chapter 37). For many years, it was assumed that ribosomal RNAs played a passive, structural role, or perhaps helped in the recognition of cognate mRNAs through a base pairing mechanism.

The RNA World Hypothesis

The discovery of ribozymes had a profound influence on evolutionary theory. For many years, scientists had hypothesized that the first biologic catalysts were formed when amino acids contained in the primordial soup coalesced to form the first simple proteins. With the realization that RNA could both carry information and catalyze simple chemical reactions, a new “RNA World” hypothesis emerged in which RNA constituted the first biological macromolecule. Eventually, DNA emerged as a more chemically stable oligonucle-otide for long-term information storage while proteins, by virtue of their much greater variety of chemical functional groups, dominated catalysis. If one assumes that some sort of RNA-protein hybrid was formed as an intermediate in the transition from ribonucleotide to polypeptide catalysts, one need look no further than the ribosome to find the presumed missing link.

Why did not proteins take over all catalytic functions? Presumably, in the case of the ribosome the process was both too complex and too essential to permit much opportunity for possible competitors to gain a foothold. In the case of the small self-cleaving RNAs and self-splicing introns, they may represent one of the few cases in which RNA autocatalysis is more efficient than development of a new protein catalyst.

SUMMARY

Image Enzymes are efficient catalysts whose stringent specificity extends to the kind of reaction catalyzed, and typically to a single substrate.

Image Organic and inorganic prosthetic groups, cofactors, and coenzymes play important roles in catalysis. Coenzymes, many of which are derivatives of B vitamins, serve as “shuttles” for commonly-used groups such as amines, electrons, and acetyl groups.

Image During catalysis, enzymes frequently redirect the conformational changes induced by substrate binding to effect complementary changes in the substrate that facilitate its transformation into product.

Image Catalytic mechanisms employed by enzymes include the introduction of strain, approximation of reactants, acid-base catalysis, and covalent catalysis. HIV protease illustrates acid-base catalysis; chymotrypsin and fructose-2,6-bisphosphatase illustrate covalent catalysis.

Image Aminoacyl residues that participate in catalysis are highly conserved among all classes of a given enzyme. Site-directed mutagenesis, used to change residues suspected of being important in catalysis or substrate binding, provides insights into mechanisms of enzyme action.

Image The catalytic activity of enzymes reveals their presence, facilitates their detection, and provides the basis for enzymelinked immunoassays. Many enzymes can be assayed spectrophotometrically by coupling them to an NAD(P)+- dependent dehydrogenase.

Image Combinatorial chemistry generates extensive libraries of potential enzyme activators and inhibitors that can be tested by high-throughput screening.

Image Assay of plasma enzymes aids diagnosis and prognosis of myocardial infarction, acute pancreatitis, and various bone and liver disorders.

Image Restriction endonucleases facilitate diagnosis of genetic diseases by revealing restriction fragment length polymorphisms, and the polymerase chain reaction (PCR) amplifies DNA initially present in quantities too small for analysis.

Image Attachment of a polyhistidyl, glutathione S-transferase (GST), or other “tag” to the N- or C-terminus of a recombinant protein facilitates its purification by affinity chromatography on a solid support that contains an immobilized ligand such as a divalent cation (eg, Ni2+) or GST. Specific proteases can then remove affinity “tags” and generate the native enzyme.

Image Not all enzymes are proteins. Several ribozymes are known that can cut and re-splice the phosphodiester bonds of RNA. In the ribosome, it is the rRNA and not the polypeptide components that are primarily responsible for catalysis.

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