Some Mechanics of Enzyme Catalysis (With Diagram)

Enzymes are proteins, and therefore their capacity for catalysis is intimately related to a specific tertiary or quaternary molecular structure.

If the tertiary or quaternary structure of an enzyme is altered, a loss of enzyme activity usually follows.

Thus, environmental factors that modify protein structure also influence enzyme activity. Key environmental factors that can affect enzyme activity are pH and temperature.

The polar side chains of certain amino acids form electrostatic bonds with each other and with surrounding ions and water molecules; these interactions contribute in part to the specific tertiary and quaternary structure of the pro­tein.

Whether a particular amino acid side chain bears a charge is determined in part by the pH of the pro­tein’s environment.

As the pH is lowered (i.e., the con­centration of H⁺ is increased), groups that may be negatively charged, such as the secondary COO^– of aspartic acid and glutamic acid and the O^– of tyro­sine, become protonated, thereby neutralizing these negative charges.

At the same time, some secondary amino groups, such as those of lysine and arginine, may accept additional protons, thereby imparting charge to these formerly neutral side chains. In con­trast, as the pH is elevated (i.e., the concentration of OH⁺ is increased), positively charged side chains dis­sociate protons and are thereby neutralized, while the loss of protons from secondary COOH and OH groups renders these groups negative. Some of these rela­tionships are depicted in Figure 8-11.

In addition to playing important roles in the mainte­nance of a specific tertiary or quaternary molecular structure, the polar side chains of some of the amino acids in the enzyme (i.e., those in the active site) may be involved in binding the substrate to the enzyme and thereby introduce bond strains into the substrate mol­ecule. Consequently, most enzymes can operate only within a narrow pH range and display a pH optimum (Fig. 8-12).

The pH optima of enzymes vary over a broad range of values. On either side of the pH optimum, enzyme activity declines as the configuration of the protein is altered and/or its affinity for the substrate is corre­spondingly decreased.

Temperature also influences enzyme activity, and most enzymes display a temperature optimum close to the normal temperature of the cell or organism pos­sessing that enzyme. Accordingly, the temperature optima of plant cell enzymes and enzymes of poikilothermic (“cold-blooded”) animals inhabiting cold re­gions of the earth are usually lower than those of en­zymes of homeothermic animals.

If the temperature is elevated far above the opti­mum, enzyme activity decreases. This is the result of an alteration of the enzyme’s structure (an unraveling process called denaturation). Most enzymes are irre­versibly denatured if maintained at temperatures above 55° to 65°C for an extended period of time.

The Active Site:

The formation of the enzyme-substrate complex is not a random process. This was recognized as long ago as 1894 when Emil Fischer postulated that an enzyme al­lows only one or a few compounds to fit onto its sur­face. This is the “lock-and-key” hypothesis according to which the enzyme and its substrate have comple­mentary shapes (see below).

The specific substrate molecules (and prosthetic groups, if any) are bound to a specific region of the enzyme molecule called its ac­tive (or catalytic) site.

The active site of an enzyme is formed by a number of amino acid residues whose side chains have two principal roles:

(1) They serve to at­tract and orient the substrate in a specific manner within the site (such amino acids are called contact residues and contribute in large degree to substrate specificity) and

(2) They participate in the formation of temporary bonds with the substrate molecule, bonds that polarize the substrate, introduce strain into cer­tain of its bonds, and trigger the catalytic change (such amino acids are termed catalytic residues).

The bonds formed between a substrate and the amino acid side chains forming the active site may be either covalent or non-covalent. The contact and catalytic residues that make up the active site may be located in widely separated regions of the enzyme’s primary structure, but as a result of stabilized polypeptide chain folding, they are brought into the appropriate juxtaposition.

For example, the active site of the enzyme citrate synthase contains about 16 amino acids; eight of these form temporary bonds with the substrate (citric acid) and another eight form bonds with a coenzyme (coenzyme A). These amino acid residues are scattered through the enzyme’s primary structure from amino acid position number 46 to position number 421. Even in relatively small enzymes such as ribonuclease, contact and cata­lytic residues are scattered over much of the primary structure (i.e., the shaded residues in Fig. 8-13).

“Lock-and-Key” versus “Induced-Fit” Models of Enzyme Action:

Figure 8-14 depicts the interaction between enzyme and substrate according to the lock-and-key model. The substrate has polar (i.e., + and -) and nonpolar (H, hydrophobic) regions and is attracted to and asso­ciates with the active site, which is complementary in both shape and charge distribution (Figs. 8-14a and 8-14b).

Positive, negative, and hydrophobic regions of the active site are created by the side chains of the contact residues, which align the substrate for inter­action with the site’s catalytic residues (A and B). Following catalysis (Fig. 8-14c), the products are re­leased from the active site (Fig. 8-14(2), thereby free­ing the enzyme for another round of catalysis. The lock-and-key model of enzyme catalysis accounts for enzyme specificity, because compounds that lack the appropriate shape or are too large or too small (Fig. 8-14e) cannot be bound to the active site.

Although the lock-and-key model accounts for much of the substrate specificity data, certain observations about enzyme behavior do not fit or are difficult to ex­plain using this model. For example, there are a num­ber of instances in which compounds other than the true substrate bind to the enzyme even though they fail to form reaction products.

Furthermore, for many enzyme-catalyzed reactions, substrates are bound to the active site in a specific temporal order. In the 1960s, Daniel Koshland proposed the “induced-fit” theory of enzyme action according to which the active site of the enzyme does not initially exist in a shape that is complementary to the substrate but is induced to assume the complementary shape as the substrate becomes bound.

As Koshland put it, the active site is induced to assume the complementary shape “in much the same way as a hand induces a change in the shape of a glove.” Thus, according to this model, the enzyme (or its active site) is flexible.

The induced-fit model is depicted diagrammatically in Figure 8-15. The active site and substrate initially have different shapes (Fig. 8-15a) but become comple­mentary on substrate binding (Fig. 8-15b). The shape change places the catalytic residues in position to al­ter the bonds in the substrate (Fig. 8-15c), following which the products are released (Fig. 8-15d) and the active site returns to its initial state.

Although mole­cules that are larger or smaller than the true sub­strate or that have different chemical properties may nonetheless be bound to the active site, none succeed in inducing the proper alignment of catalytic groups, and no catalysis occurs (Figs. 8-15e and 8-15f). The induced-fit model explains the effects of certain com­petitive and noncompetitive inhibitors of enzyme action.

Before proceeding further, it should be acknowl­edged that some enzyme-catalyzed reactions are adequately explained by the lock-and-key model, so that a flexible active site is not a strict requirement for catalysis. By the same token, the possession of a flexible active site does not imply that any molecule may become bound to the enzyme.

A change in the shape of the active site of an en­zyme can also be induced by binding of substances at sites on the enzyme’s surface that are far removed from the active site. In such a case, the change is transmitted through the enzyme molecule from the site of binding to the active site. Such changes may ei­ther decrease or increase the enzyme’s activity.