Chemistry and Biology of Proteins - F. Haurowitz 1953

Proteins with Enzymatic Properties
Mechanism of Enzymatic Hydrolysis

The second phase of Enzymatic Hydrolysis is the hydrolytic Cleavage of the substrate AB into AOH and BH. Since no appreciable hydrolytic cleavage of AB occurs in the absence of the enzyme, it must be assumed that it is not AB itself that undergoes cleavage, but rather the E(AB) complex. Within this complex, the bonds between A and B are weakened, which facilitates hydrolytic breakdown. The exact mechanism by which the bonds between A and B are loosened remains to be fully elucidated. The most plausible hypothesis is that the binding of the substrate molecule to the enzyme induces a deformation in the substrate, and either through mechanical forces or the electrostatic influence of neighboring polar groups, the bonds of AB are ruptured. Such molecules are referred to as activated.

By supplying external energy (such as thermal energy), AB molecules can be driven into an activated state, denoted as AB*. The energy required to bring molecules into this activated state is known as the activation energy. As pointed out in Chapter VII, the activation energy ∆Н* is equal to ∆F* + Т∆S*, where ∆F* is the Standard Free energy of activation, Т is the absolute Temperature, and ∆S* is the Entropy of activation. Assuming that the dissociation of the enzyme-substrate complex into the free enzyme and reaction products occurs very rapidly, The rate of the catalytic reaction will depend primarily on the rate of the activation process. In turn, the rate of the activation process depends on the magnitude of the Free energy of activation ∆F* (see p. 164). The free energy of activation required for the acid-catalyzed hydrolysis of esters by hydrogen ions is approximately 10,000–13,000 cal/mol; for the hydrolysis of peptide bonds, it is about 20,000 cal/mol. In enzymatic reactions, the activation energy is reduced to roughly 4,000 cal/mol for ester hydrolysis and to 12,000–14,000 cal/mol for peptide hydrolysis [41]. This enzymatic effect can be expressed by the following equations:

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From a thermodynamic standpoint, Enzymes and other catalysts can be defined as substances that increase the rates of Chemical Reactions, or render such reactions possible, by lowering the activation free energy. Like all Principles of Thermodynamics, this definition concerns only the Energy balance of reactions and evaluates their thermodynamic feasibility; however, it provides no insight into the actual mechanism of enzymatic reactions—that is, The Nature of the reacting groups of the enzyme and substrate, or the character of the reaction intermediates.

To gain some understanding of The Mechanism of enzymatic reactions, one must turn to The Study of so-called enzyme models, namely, simpler substances that exhibit catalytic activity similar to that of enzymes.

Until recently, strong acids and bases were considered the simplest catalysts for hydrolysis. The action of these substances was attributed to the hydrolyzing effect of high concentrations of hydrogen or hydroxyl ions. Most hydrolytic enzymes exhibit an optimum pH of around 7—that is, under conditions where the concentrations of both hydrogen and hydroxyl ions are extremely low. This indicates that the mechanism of enzymatic hydrolysis presumably differs from that of hydrolysis by strong acids or bases. In recent years, however, a new group of hydrolytic catalysts has been discovered. Steinhardt [42] found that various organic sulfonic acids, such as dodecyl sulfonate or Orange II, possess The ability to hydrolyze amide groups even at low sulfonic acid concentrations:

RCONH₂→ RCOOH + NH₃.

Similarly, Proteins undergo hydrolysis in the presence of 0.1–0.01 M dodecyl sulfonate solutions at 65°, whereas the Hydrolysis of Proteins using hydrochloric or sulfuric acid typically requires significantly higher concentrations [43]. It should be noted that the hydrolysis of proteins by sulfonic acids does not go to completion and halts when approximately 50% of The amino acid residues have been cleaved [43].

Catalytic activity is exhibited not only by Water-soluble sulfonic acids but also by insoluble resins containing sulfonic acid groups. For example, Amberlite IR 100 (a resin containing 1 g-equiv of SO₃H per 500 g of substance) hydrolyzes ethyl acetate at 25° [44], while a phenol-formaldehydesulfonic acid resin catalyzes the synthesis of an ester from oleic acid and butyl alcohol [45]. Evidently, the sulfonic acid group in Organic compounds displays a higher catalytic activity than sulfuric acid at the same concentration. Since the dissociation constants of organic acids are lower than that of sulfuric acid, there is no reason to assume that the catalytic activity of sulfonic acids is due to a higher hydrogen ion concentration compared to sulfuric acid. The activity of sulfonic acids evidently depends on The Structure of the organic moiety of the sulfonic acid molecule. It may be hypothesized that the organic backbone acts in a sense as an apoenzyme, whereas the sulfonic acid group performs the function of a coenzyme.

However, all the observations cited above can be interpreted in an entirely different manner—namely, one might assume that the catalytic activity of the enzyme depends on the clustering of A large number of ionic groups within a restricted space of the enzyme protein molecule. It is well known (though this phenomenon remains unexplained to this day) that the activity of ions in concentrated solutions of alkalis or mineral salts exceeds the theoretical value by more than 100 times. By analogy, one can imagine that the aggregation of a large number of polar groups in a small region of a protein molecule would result in unusually high activity and, consequently, high catalytic efficiency. Indeed, it has been found that tetravalent lanthanum ions are capable of catalyzing the hydrolysis of meta- or pyrophosphates to orthophosphate [46]. As noted above, many peptidases [16] and certain Phosphatases [17] contain Metal Ions, such as Mg⁺⁺ or Mn⁺⁺. In the enzyme-substrate com

plex, the linkage between the polar groups of the enzyme and those of the peptide is mediated through metal ions. Through this form of bonding, ring structures can be formed in which the electronic configuration of the peptide bond is distorted to such an extent that the hydrolytic process is initiated [15, 16, 26, 47].

The METABOLISM/10.html">Mechanism of enzyme-substrate complex formation is clearly illustrated by Reactions Catalyzed by phosphomutases or Kinases. One such enzyme is phosphoglucomutase, which catalyzes the equilibrium between glucose-1-phosphate and glucose-6-phosphate. The enzyme contains tightly bound phosphorus that cannot be removed by dialysis. However, when glucose-1-phosphate containing radioactive phosphorus is added to the enzyme, an exchange of phosphorus atoms takes place between the enzyme and the glucose-1-phosphate [48]. This reaction can be represented schematically as follows [49, 50]¹:

As seen from the given formulas, the phosphate residue is transferred by the enzyme from the first carbon atom of glucose-1,6-diphosphate to the sixth carbon atom of glucose-1-phosphate, and from the sixth carbon atom of the diphosphate to the first carbon atom of glucose-6-phosphate. This has been proven using ¹⁴C-labeled glucose phosphates. Obviously, the reaction can proceed only in the presence of glucose-1,6-diphosphate; since the diphosphate is continuously regenerated, only trace amounts of this substance are required for the reaction [49, 50]. Similar results were obtained with phosphoglyceromutase, which catalyzes the interconversion of 2-phosphoglyceric acid and 3-phosphoglyceric acid [50]. In both cases, the action of the enzyme consists in temporarily binding to a phosphoric acid residue and transferring it from one molecule to another. Myokinase and hexokinase act in a similar fashion as phosphotransferases (see below). If the phosphate residue is transferred to a water molecule, hydrolysis of the organic phosphorus compound ensues. Accordingly, phosphatases can be viewed as phosphotransferases that catalyze The transfer of a phosphate group from organic compounds to water, or from inorganic phosphate to organic compounds. Similarly, Proteolytic Enzymes can be regarded as aminoacyltransferases which, upon binding to an amino acid residue, transfer it to another amino acid, a peptide, or a water molecule. If the amino acid residue is transferred to another amino acid, new Peptides are formed; if it is transferred to a water molecule, hydrolysis takes place with the liberation of free Amino Acids.

¹ In the English edition of the book, the author made errors in describing the Transfer of phosphate residues from glucose-1,6-diphosphate to glucose-1-phosphate and glucose phosphate, both in the structural formulas depicting this process and in the subsequent explanatory text. During the Introduction/27.html">Translation of the book, these errors were corrected, and the phosphate transfer scheme and explanatory text are given in the form presented in the original work by E. W. Sutherland, M. Cohn, T. Posternak, and C. Cori (J. Biol. Chem., 180, 1285, 1949), cited by the author of this book. — Ed. note.

From all the foregoing, it follows that the binding of an enzyme to its substrate depends primarily on the configuration and spatial arrangement of the reactive groups of both the enzyme and the substrate. If the reactive group of the enzyme can come into close contact with the reactive group of the substrate, an enzyme-substrate complex is formed and the catalytic reaction proceeds.

To date, very little is known about the reactive groups of Hydrolases. Certain insights regarding the reactive groups of their substrates can be gained by studying the rates of catalytic reactions in the presence of Competitive Inhibitors [51]. Competitive inhibitors are substances that structurally resemble the substrates of hydrolytic enzymes but differ in certain molecular features. Possessing the ability to bind to the enzyme just like the substrate, they compete with it and thereby reduce the rate of the catalytic reaction.

Experiments of this kind have demonstrated that the aromatic ring of Tyrosine or phenylalanine is essential for the action of Chymotrypsin or carboxypeptidase on Polypeptides [20, 52]. Evidently, the electronic System of the aromatic ring participates in the binding reaction of chymotrypsin or carboxypeptidase with their substrates. Since the benzene ring is neither ionized nor dipolar, there is no reason to suppose that it binds to the polar groups of the enzyme. It is possible that the aromatic ring of the substrate associates with a structurally similar aromatic ring on the enzyme.

It is evident from the foregoing that although many details concerning the mechanism of enzyme-substrate complex formation remain unclear, it is beyond doubt that this process involves multiple reactive groups on both the substrate and the enzyme. This is consistent with data on the high Specificity of enzymatic reactions and Supports the concept that the surface geometries of the reacting groups of the enzyme and substrate are mutually complementary.