General Base, Nucleophilic Catalysis a-Chymotrypsin

Chymotrypsin catalysis takes place through a three-step process, equation (11), where ES is an enzyme substrate complex which breaks down to give an acylated enzyme intermediate, ES and Pj, 

Bruice and Sturtevant, (1959) and Bruice, (1959) found extremely facile intramolecular nucleophilic attack by neighbouring imidazole in the hydrolysis of p-nitrophenyl 7-(4-imidazoyl)butyrate [19]. The rate constant for imidazole participation (release of p-nitro-phenolate) in this reaction is nearly identical with the rate constant for a-chymotrypsin catalysed release of p-nitrophenolate ion [190 min in equation (11) at pH 7 and 25°] from p-nitrophenyl acetate. Comparison of the rate constant for intramolecular imidazole participation to that for the analogous bimolecular reaction (imidazole attack on p-nitrophenyl acetate) (s /m s ) 

Imidazole will function as a general base in the hydrolysis of acyl-activated esters such as ethyl dichloroacetate Qencks and Carriuolo, 1961) and esters where the p/STg-value of the leaving group is 2-3 units lower than that of ethanol and methanol such as 

2-dichIoroethyl acetate (Bruice et al., 1962a) or N,0-diacetyl-serinamide (Anderson et al., 1961 Milstien and Fife, 1968). The mechanism in these examples involves proton transfer in the critical transition state as shown by ratios of 2-3. Thus, the 

Intramolecular general base catalysis of hycholysis (21a) was unexpected since the ester has a phenolic leaving group. Felton and Bruice (1968, 1969) reasoned that, if nucleophilic attack occurred, the leaving phenolate ion group would be properly positioned to attack the intermediate acylimidazole and thereby reverse the reaction. The normally less efficient general base reaction then becomes the favoured pathway, as in hydrolysis of acetyl salicylate (see Section 4). Likewise, Fife and McMahon (1970) explained bimolecular general base catalysis by imidazole (21b) in hydrolysis of o-(4-nitrophenylene) carbonate 3 49) by reversibility 

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In chymotrypsin and other serine proteases the imidazole moiety of histidine acts as a general base not as a nucleophile as is probably the case in the catalysis of activated phenyl ester hydrolysis by (26). With this idea in mind, Kiefer et al. 40) studied the hydrolysis of 4-nitrocatechol sulfate in the presence of (26) since aryl sulfatase, the corresponding enzyme, has imidazole at the active center. Dramatic results were obtained. The substrate, nitrocatechol sulfate, is very stable in water at room temperature. Even the presence of 2M imidazole does not produce detectable hydrolysis. In contrast (26) cleaves the substrate at 20°C. Michaelis-Menten kinetics were obtained the second-order rate constant for catalysis by (26) is 10 times... 

Considerable effort has been applied to studies of ester hydrolysis catalyzed by imidazoles (76MI40700, 80AHC(27)241). Certainly, 1-acetylimidazole can be made enzymically, probably by the sequence acetyl phosphate + coenzyme A acetylcoenzyme A+phosphate, acetyl-coenzyme A + imidazole l-acetylimidazole+coenzyme A. In addition, the imidazolyl group of histidine appears to be implicated in the mode of action of such hydrolytic enzymes as trypsin and chymotrypsin, thereby engendering further interest in the process of imidazole catalysis. The two pathways which have been found to be involved are general base catalysis and nucleophilic catalysis. In the former (Scheme 26) a basic imidazole molecule can activate a water molecule to attack the ester at the carbonyl carbon, this being followed by the usual sequence of steps as in simple hydroxide ion hydrolysis. At high imidazole concentrations the imidazole molecules may be involved directly. 

General acid-base catalysis. In general acid-base catalysis, a molecule other than water plays the role of a proton donor or acceptor. Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine (Section 9.1.3). 

Figure 37.2. Catalysis by the enzyme chymotrypsin of the cleavage of one peptide bond in a protein a proposed mechanism. Histidine and pro-tonated histidine act as general base and acid in two successive nucleophilic substitution reactions (a) cleavage of protein with formation of acyl enzyme and liberation of one protein fragment (6) hydrolysis of acyl enzyme with regeneration of the enzyme and liberation of the other protein fragment.

FIGURE 6-10 Covalent and general acid-base catalysis. The first step in the reaction catalyzed by chymotrypsin is the acylation step. The hydroxyl group of Ser is the nucleophile in a reaction aided by general base catalysis (the base is the side chain of His ). This provides a new pathway for the hydrolytic cleavage of a peptide bond. Catalysis occurs only if each step in the new pathway is faster than the uncatalyzed reaction. The chymotrypsin reaction is described in more detail in Figure 5-21. 

The above-considered calculational data point to high effectiveness of the bifunctional catalysis in hydrolytic reactions and the reactions related to these, due to involvement of molecular chains of water and ammonia, as well as to the preferability in these reactions of a concerted mechanism. This conclusion is fairly general and is corroborated by calculations on other types of nucleophilic reactions, such as hydrolysis of methyl fluoride, tautomerization of pyridine in aqueous solution etc. [110]. An advisable piece of work would apparently, be an analysis, in the light of the conclusions discussed, of mechanisms of the catalytic act in enzymic hydrolysis reactions of the ester and peptide bonds. In the most advanced up-to-date models for, e.g., the reactions with participation of a-chymotrypsin (see Ref. [Ill]), the steps of the base and the acid catalysis are separated. The latter is commonly thought [84, 111] to be operative at the stage of enzymic decomposition of the tetrahedral intermediate. However, taking into account the possibility of realization of the conformationally excited states of the active enzymic center, it would not be hard to think of some realistic schemes of concerted mechanisms, the more so that the fast growing body of calculational material continuously supplies fresh evidence in favor of such mechanisms. 

Enzymes may use any of the above mentioned modes of catalysis in order to catalyze a particular chemical reaction. For example, the imidazole ring of a histidine residue of the enzyme a-chymotrypsin (Section 4.4) can function as a general-base catalyst, while in the enzyme alkaline phosphatase, the same residue can function as a nucleophilic catalyst. Indeed, enzymes are complex catalysts which employ more than one catalytic parameter during the course of their action. It is by this successful integration of a combination of individual catalytic processes that a rate enhancement as high as 10 " may be achieved. Furthermore, it is this combination of factors which results in a specific catalyst. 

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