Chemistry and Biology of Proteins - F. Haurowitz 1953

Proteins with Enzymatic Properties
Combination of an enzyme with a substrate (enzyme-substrate complex)

Although the Mechanism of Enzymatic action cannot be explained solely by The formation of intermediate compounds between the enzyme and the substrate for reasons that will be discussed below, we must nevertheless accept that The first phase of an enzymatic reaction is the formation of an enzyme-substrate complex. At the same time, it must also be assumed that the resulting enzyme-substrate complexes are unstable and can exist only for a short period of time. If the enzyme formed very stable complexes with the substrate, the entire amount of the enzyme would eventually become bound to the substrate, and the latter would not undergo any further transformation.

Our views on Chemical Reactions and catalysis are grounded in the concept that a chemical reaction can occur only As a result of collisions between reacting molecules. According to this premise, substances A and B cannot react to form C or D unless an AB complex is formed, at least temporarily, during the reaction.

Although this view is widely accepted, it is contradicted by reports indicating that Trypsin can split serum albumin even when the latter is separated from it by a film thicker than 100 Å [27]. Based on these experiments, it was concluded that Enzymes are capable of acting on a substrate located at a certain distance from them. It should be noted, however, that there are currently no sufficiently precise Methods to completely rule out the inhomogeneity of the film [28], and consequently, the possibility of the substrate or enzyme penetrating through it. Therefore, the Conclusions of these studies cannot be considered sufficiently substantiated. It is hardly necessary to emphasize that the law of mass action, as well as other fundamental laws of chemical kinetics, is based on the acceptance of the collision concept between reacting molecules. The idea of reactions taking place between molecules that do not come into contact with each other is completely incompatible with the numerous facts supporting the molecular-kinetic theory of chemical reactions.

The formation of an enzyme-substrate complex in the initial phase of a reaction was first confirmed by studying reaction kinetics in the presence of substances that have The ability to be preferentially bound by the enzyme, competing in this respect with the substrate. Two groups of such substances are currently known: reaction products and specific enzyme poisons.

The inhibitory effect of the products of enzymatic catalytic activity on The activity of the enzymes themselves can be demonstrated using hydrolytic enzymes as an example [29–31]. Let us consider the hydrolytic Cleavage of substance AB:

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and designate the enzyme-substrate complex as E(AB), where E is the free enzyme. If Hydrolysis is inhibited by The addition of an excess of AOH, but not BH, then it can be stated with certainty that the enzyme binds to component A of the substrate AB rather than to component B. By this method, it was found that the hydrolysis of sucrose by Yeast invertase is inhibited by fructose and not inhibited by glucose. From this, it can be concluded that the enzyme binds to the fructose moiety of sucrose [29, 31]. The first phase of this enzymatic reaction can therefore be represented by the following equation:

where E is the enzyme molecule, A is the fructose component of sucrose, and B is the glucose component of sucrose [29, 31].

The binding of the enzyme to the substrate is also inhibited by other substances capable of forming stable compounds with the enzyme. A well-known example is the inhibitory effect of cyanides on the action of enzymes containing a heme group as their Active Site. It is known that hydrocyanic acid forms a stable complex with the iron atom of the heme, thereby depriving the iron atom of the ability to bind to substrate molecules.

Until relatively recently, the hypothesis regarding the formation of intermediate compounds between the enzyme and the substrate was based primarily on kinetic data obtained under various conditions. Only very recently has it been possible to prove spectrophotometrically the formation of complexes with substrates by peroxidase and catalase. Catalase and peroxidase are colored compounds possessing a characteristic absorption spectrum. When these enzymes form intermediate compounds with substrates, characteristic changes occur in both color and absorption spectrum. Initially, a green complex is formed, which then transforms into a red complex [32]. Measurements of the rates of these transformations have shown that the Formation of the green complex occurs very rapidly (k₁≈ 10⁷), whereas The conversion of the green complex into the red one proceeds slowly (k₂ = 4.0 for peroxidase). Furthermore, it was shown that only the green complex serves as the intermediate compound in the reaction between the enzyme and the substrate. This green complex slowly dissociates into the free enzyme and reaction products [33]. In all likelihood, the iron incorporated into the heme of catalase and peroxidase is linked to the protein moiety in the reactive green complex via an ionic bond, and in the red complex via covalent bonds [32, 33]. If we designate the enzyme by — the trivalent iron ion of the heme — the formation of the enzyme-hydrogen peroxide complex can be represented by the following equation [32, 33]: