Proteins that have catalytic properties are called enzymes (i.e., enzymes are biological catalysts of protein nature). Some enzymes have full catalytic reactivity per se; these are considered simple proteins because they do not have a nonprotein moiety. Other enzymes are conjugated proteins, and the nonprotein structural components are necessary for reactivity. Occasionally, enzymes require metallic ions. Because enzymes are proteins or conjugated proteins, the general review of protein structural studies presented previously in this chapter (e.g., protein conformation and denaturation) is fundamental to the following topics. Conditions that affect denaturation of proteins usually have an adverse effect on the activity of the enzyme.
General enzymology is discussed effectively in numerous standard treatises, and one of the most concise discussions appears in the classic work by Ferdinand,14 who includes reviews of enzyme structure and function, bioenergetics, and kinetics and appropriate illustrations with a total of 37 enzymes selected from the six major classes. For additional basic studies of enzymology, the reader should refer to this classic monograph and to a comprehensive review of this topic.15
Koshland16 has reviewed concepts concerning correlations of protein conformation and conformational flexibility of enzymes with enzyme catalysis. Enzymes do not exist initially in a conformation complementary to that of the substrate. The substrate induces the enzyme to assume a complementary conformation. This is the so-called induced-fit theory. There is proof that proteins do possess conforma-tional flexibility and undergo conformational changes under the influence of small molecules. This does not mean that all proteins must be flexible; nor does it mean that conforma-tionally flexible enzymes must undergo conformational changes when interacting with all compounds. Furthermore, a regulatory compound that is not directly involved in the reaction can exert control on the reactivity of the enzyme by inducing conformational changes (i.e., by inducing the enzyme to assume the specific conformation complementary to the substrate). (Conceivably, hormones as regulators function according to the foregoing mechanism of affecting protein structure.) So-called flexible enzymes can be distorted conformationally by molecules classically called inhibitors. Such inhibitors can induce the protein to undergo conformational changes, disrupting the catalytic functions or the binding function of the enzyme. In this connection, it is noteworthy how the work of Belleau17 and the molecular perturbation theory of drug action relate to Koshland's studies presented previously in this textbook.
Evidence continues to support the explanation of enzyme catalysis based on the active site (reactive center) of amino acid residues, which is considered to be that relatively small region of the enzyme's macromolecular surface involved in catalysis. Within this site, the enzyme has strategically positioned functional groups (from the side chains of amino acid units) that participate cooperatively in the catalytic action.18
Some enzymes have absolute specificity for a single substrate; others catalyze a particular type of reaction that various compounds undergo. In the latter, the enzyme is said to have relative specificity. Nevertheless, compared with other catalysts, enzymes are outstanding in their specificity for certain substrates.19 The physical, chemical, conformational, and configurational properties of the substrate determine its complementarity to the enzyme's reactive center. These factors, therefore, determine whether a given compound satisfies the specificity of a particular enzyme. Enzyme specificity must be a function of the nature, including conformational and chemical reactivity, of the reactive center, but when the enzyme is a conjugated protein with a coenzyme moiety, the nature of the coen-zyme also contributes to specificity characteristics.
In some instances, the active center of the enzyme is apparently complementary to the substrate molecule in a strained configuration, corresponding to the "activated" complex for the reaction catalyzed by the enzyme. The substrate molecule is attracted to the enzyme, and the forces of attraction cause it to assume the strained state, with confor-mational changes that favor the chemical reaction; that is, the enzyme decreases the activation energy requirement of the reaction to such an extent that the reaction proceeds appreciably faster than it would in the absence of the enzyme. If enzymes were always completely complementary in structure to the substrates, then no other molecule would be expected to compete successfully with the substrate in combination with the enzyme, which, in this respect, would be similar in behavior to antibodies. Occasionally, however, an enzyme complementary to a strained substrate molecule attracts a molecule resembling the strained substrate molecule more strongly; for example, the hydrolysis of benzoyl-l-tyrosylglycineamide is practically inhibited by an equal amount of benzoyl-d-tyrosylglycineamide. This example illustrates a type of antimetabolite activity.
Several types of interaction contribute to the formation of enzyme-substrate complexes: attractions between charged (ionic) groups on the protein and the substrate, hydrogen bonding, hydrophobic forces (the tendency of hydrocarbon moieties of side chains of amino acid residues to associate with the nonpolar groups of the substrate in a water environment), and London forces (induced dipole interactions).
Many studies of enzyme specificity have involved pro-teolytic enzymes (proteases). Configurational specificity can be exemplified by the aminopeptidase that cleaves l-leucylglycylglycine but does not affect d-leucylglycyl-glycine. d-Alanylglycylglycine is cleaved slowly by this enzyme. These phenomena illustrate the significance of steric factors; at the active center of aminopeptidase, the closeness of approach affects the kinetics of the reaction.
One can easily imagine how difficult it is to study the reactivity of enzymes on a functional group basis because the mechanism of enzyme action is so complex.16 Nevertheless, the —SH group probably is found in more enzymes as a functional group than are the other polar groups. In some enzymes (e.g., urease), the less readily available SH groups are necessary for biological activity and cannot be detected by the nitroprusside test, which is used to detect freely reactive SH groups.
A free —OH group of the tyrosyl residue is necessary for the activity of pepsin. Both the —OH of serine and the imidazole portion of histidine appear to be necessary parts of the active center of certain hydrolytic enzymes, such as trypsin and chymotrypsin, and furnish the electrostatic forces involved in a proposed mechanism (Fig. 27.3), in which E denotes enzyme and the other symbols are self-evident. (Alternative mechanisms have been proposed15; es-terification and hydrolysis were studied extensively by M. L. Bender.19a-d D. M. Blow reviewed studies concerning the structure and mechanism of chymotrypsin.19e )
These two groups (i.e., —OH and =NH) could be located on separate peptide chains in the enzyme as long as the specific three-dimensional structure formed during activation of the zymogen brought them near enough to form a hydrogen bond. The polarization of the resulting structure would cause the serine oxygen to be the nucleophilic agent that attacks the carbonyl function of the substrate. The complex is stabilized by the simultaneous "exchange" of the hydrogen bond from the serine oxygen to the carbonyl oxygen of the substrate.
Stable (Intermediate) Acyl Enzyme
Stable (Intermediate) Acyl Enzyme
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