Chymotrypsin ester hydrolysis

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... 

TETRAHEDRAL INTERMEDIATE ESTER HYDROLYSIS MECHANISM CHYMOTRYPSIN SERPINS (Inhibitory Mechanism)... 

Fig. 5. Reaction mechanism for carboxyl ester hydrolysis by chymotrypsin.

One of the most investigated type of reaction in the field of catalytic imprinted polymers, as indicated by the large number of publications available, is certainly ester hydrolysis. In particular, a great deal of work has been carried out on systems inspired by hydrolytic enzymes since 1987. In 2000, Shea et al. [37] reported the preparation of enantioselective imprinted polymers for the hydrolysis of N-tert-butoxycarbonyl phenylalanine-p-nitrophenyl ester (55), using a system already developed by the same group in 1994 [19]. The system was inspired by the natural hydrolytic enzyme chymotrypsin and polymerisable imidazole units (27) were used as functional monomers coupled via ester linkages to a chiral phosphonate (56), analogue of (d)- or (L)-phenyl-alanine. After template removal, the imprinted polymers showed selectivity towards the hydrolysis of the enantiomer with which they were imprinted. The ratio of the rate constants, k /k, was 1.9 for the polymer imprinted with the D-enantiomer and kjku was 1.2 for that imprinted with the L-enantiomer. Moreover, the imprinted polymer showed a 2.5-fold increase in the rate of the reaction when compared with the control polymer, imprinted with a... 

Kinetic studies of a-chymotrypsin catalyzed hydrolysis of p-nitrophenyl esters are carried out (Duprix et al., 1970) ... 

Perform the regression analyses for the descriptors to assess the contribution of substituent effect(s) on the rate of a-chymotrypsin-catalyzed hydrolysis of p-nit-rophenyl esters. Referring to the catalytic triad of chymotrypsin, rationalize your results for the plausible reaction mechanism. 

With a polyethylenimine containing 10% of its residues alkylated with dodecyl groups and 15 % alkylated with methyleneimidazole substituents, esterolysis is truly catalytic [16]. Table 3.2 compares the catalytic effectiveness of this polymer biocatalyst (synzyme) with that reported for other substances that accentuate nitrophenyl ester hydrolysis [17, 18]. Clearly, this polymer is nearly 300 times as effective as free imidazole, but it does not match chymotrypsin, even with the activated unnatural nitrophenyl ester substrate, let alone peptide substrates. 

Reaction course of the ester hydrolysis by a-chymotrypsin is written as follows,... 

The effect of added co-solvent on the initial state is also important in more complicated reactions. For example, in the a-chymotrypsin-catalysed hydrolysis of p-nitrophenyl acetate and of N-acetyl-L-tryptophan methyl ester, the difference in the pattern of rates of hydrolysis when the solvent composition is varied can be attributed to the variation in the properties of the initial states of the esters (Bell et al., 1974). 

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. 

The specificity of chymotrypsin for hydrolysis of peptide bonds formed by the carbo,xyl groups of tyrosine, phenylalanine, and tryptophan has been recognized for some time (Green and Neurath, 1954 Desnuelle, 1960). Action on synthetic substrates of leucine (Goldenberg et al., 1951) and methionine (Kaufman and Neurath, 1949) also has been noted although at much slower rates than observed with the aromatic amino acid derivatives. When protein substrates or synthetic ester substrates are examined, it is evident that a variety of bonds can be hydrolyzed by chymotrypsin. Inagami and Sturtevant (1960) observed that chymotryptic hydrolysis of a-benzoyl-L-arginine ethyl ester, a typical trypsin substrate, occurred at a maximum rate which was 20% of that observed with trypsin. Several ester substrates, such as p-nitrophenylacetate (Hartley and Kilby, 1954), are also hydrolyzed. 

This intermediate is formed during the a-chymotrypsin-catalyzed hydrolysis of esters, by the following reaction scheme ... 

Examples of enantioselective hydrolysis of cyclic diesters by a-chymotrypsin are comparatively rare (10-14) (Table 11.1-7). Interestingly, the cyclopentanoid and the cyclohexenoid monoesters 11 and 12 have the opposite absolute configuration to those obtained by the pig liver esterase-catalyzed hydrolysis of the corresponding diesters (Table 11.1-1). The keto ester 14, which is a valuable building block for the synthesis of prostacyclin analogs, has been obtained from the corresponding a,a -keto diester via a-chymotrypsin-catalyzed hydrolysis followed by a decarboxylation of the keto acid. 

As with peptide hydrolysis, several enzyme systems exist that catalyze carboxylic and phosphoric ester hydrolysis without the need for a metal ion. They generally involve a serine residue as the nucleophile in turn, serine may be activated by hydrogen-bond formation—or even proton abstraction—by other acid-base groups in the active site. The reaction proceeds to form an acyl- or phosphory 1-enzyme intermediate, which is then hydrolyzed with readdition of a proton to the serine oxygen. Mechanisms of this type have been proposed for chymotrypsin. In glucose-6-phosphatase the nucleophile has been proposed to be a histidine residue. ... 

We have shown by a comparison of the pH dependence of the step characterized by ki that the hydrolysis of the enzyme-acyl compound is the rate-determining step for the enzymatic hydrolysis of the usual amino acid amide substrates. In the case of chymotrypsin, acetyl-L-phenylalanine ethyl ester is hydrolyzed 1,000 times faster than the corresponding amide and in the case of trypsin, benzoyl-L-arginine ethyl ester is hydrolyzed 300 times faster than the corresponding amide. This suggests that for the amide hydrolysis too the second step, the acylation of the enzyme, must be the rate-determining step, since the third step is obviously identical for esters and amides of the same amino acid derivatives. The pH dependence of the chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester and acetyl-L-phenylalanine ethyl ester indicates that for these reactions ki and kz are of the same order of magnitude and both contribute to the over-all rate, as shown by Equation (4). 

A hydrophilic substrate, acetylsalicylic acid, was subjected to lipase catalyzed hydrolysis in a W/O microemulsion [77]. For comparison, the reaction was also carried out in aqueous buffer. Since hydrolysis of acetylsalicylic acid proceeds spontaneously without added catalyst (intramolecular catalysis), reactions without lipase were performed as controls. It was found that addition of lipase did not affect the rate of reaction in aqueous buffer. However, the reaction in miroemulsion was catalyzed by the lipase, and the rate was linearly dependent on lipase concentration. This is a further illustration of the fact that microemulsions, with their large oil/water interfaces, are suitable media for lipase-catalyzed reactions. The same reactions were also performed using a-chymotrypsin as catalyst. This enzyme, which also catalyzes ester hydrolysis but which, unlike lipase, functions independently of a hydrophobic surface, was not more active in microemulsion than in the buffer solution. 

This is the only example of the cyclodextrin-catalyzed hydrolysis of an amide. The rate-determining step in this process is acylation whereas the rate-determining step in the cyclodextrin-catalyzed hydrolysis of phenyl esters is deacylation. This dichotomy completely parallels the situation in chymotrypsin-catalyzed hydrolysis as shown in Table 3. 

Scheme 2.50 Mirror-image oiicaitation of the eatalytie machinery of Candida rugosa lipase and the protease subtilisin and enantiocomplcanentary ester hydrolysis using Mucor sp. lipase and a-chymotrypsin...

CD has been extensively studied for hydrolysis of phenyl ester as a model of serine protease, because CD has a hydrophobic cavity which acts like a binding site of enzyme [1]. These studies show that CD-catalyzed hydrolysis proceeds quite similarly to enzyme hydrolysis. However, there are three differences between CD-catalyzed hydrolysis and chymotrypsin-catalyzed hydrolysis. 

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