Chymotrypsin deacylation

These results were mechanistically interesting since the 20 20 of cinnamoyl-chymotrypsin deacylation = 2.5. It was so mechanistically interesting that we decided to compare rates. To our horror we found that the enzjnne deacylation was still about a thousand-fold faster than our model. In analyzing our data there was obviously one group missing, a carboxylate ion. To correct this discrepancy, we added benzoate ion,... 

Figure 4.34b. Second stage in the hydrolysis of a peptide by chymotrypsin deacylation. The acyl-enzyme intermediate is hydrolyzed by water. Note that deacylation is essentially the reverse of acylation, with water in the role of the amine component of the original substrate, adapted from L. Stryerl .

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

Fig. 21 Rate-pH dependence of the acylation and deacylation steps in the chymotrypsin-catalysed hydrolysis of 4-nitrophenyl trimethylacetate...

Kirsh et al. 42) prepared apolar derivatives of poly(4-vinylpyridine) by benzylation. With nitrophenyl acetate as the substrate the benzylated catalyst is 100 times more effective than 4-ethylpyridine. A double-displacement mechanism was observed. The rate constants for deacylation of the acylpoly(vinylpyridine) derivatives were about 4 x 10" /sec. The comparable value for a-chymotrypsin is 8 x 10 /sec. The factor of 20 seems small, but it should be kept in mind that deacetylation of a-chymotrypsin is very slow compared with the deacylation reactions involving the natural substrates of the enzyme. 

There is now convincing evidence that an acyl chymotrypsin intermediate is formed from both specific and non-specific substrates (Bender and Kezdy, 1964 Bender et al., 1964). This intermediate is undoubtedly an acylserine. Acyl- and phosphorylserine derivatives have been isolated and identified. In view of evidence such as a D2 O solvent isotope effect ( h2oAd2o) 2-3 for both acylation and deacylation (Bender and Hamilton, 1962), alcohol and amine nucleophiles showing little dependence on the p/iTa-value of the nucleophile in reaction with furoyl enzyme (Inward and Jencks, 1965), and the influence of increasing steric bulk in the acyl group (Fife and Milstien, 1967 Milstien and Fife, 1968,.1969), consistent... 

All of this evidence supports the existence of tetrahedral intermediates in a-chymotrypsin-catalysed reactions, but it should be noted that O-exchange with water is not observed in deacylation of cinnamoyl- 0-chymotrypsin, in contrast with the hydrolysis of O-cinnamoyl-N-acetylserinamide where such exchange is detected (Bender and Heck, 1967). Lack of exchange in the enzyme reaction could reflect interactions of the tetrahedral intermediate with the protein. 

In accord with the above scheme, variation of the meta-substituent altered the rate of acylation but not that of deacylation. The reaction of equation (27) is similar to reaction of a-chymotrypsin with phenolic esters. 

In like manner, increased steric bulk in the acyl group of acyl-a-chymotrypsin progressively retards the rate of deacylation [6 in the... 

The shapes of the curves in Fig. 6 are consistent with a two-step pathway, analogous to that of a hydrolytic enzyme such as a-chymotrypsin,30 in which an initial acylation burst is followed by a slow deacylation reaction. Following a fast preequilibrium binding, the first kinetic step can be attributed to acylation by substrate of the polymer imidazole residue, accompanied by simultaneous release of nit-rophenol(ate). The succeeding kinetic step would then be ascribed to hydrolysis of the acylimidazole leading to carboxylate ion and regenerated imidazole. 

The reaction of 14 may remind one of the well-established reaction mechanism for chymotrypsin (Fig. 5) (20). By comparing the acyl-trans-fer reaction of complex 14 with that of chymotrypsin 17, we find that the alcoholic nucleophiles in 14 and 17 are activated by Zn11—OH- and imidazole (in a triad), respectively. Several common features should be pointed out (i) Both reactions proceed via two-step reaction (i.e., double displacement), (ii) The basicity of Zn11—OH (pKa = 7.7) is somewhat similar to that of imidazole (plfa = ca. 7). (iii) The initial acyl-transfer reactions to alcoholic OH groups are rate determining, (iv) In NA hydrolysis with chymotrypsin, the pH dependence of both the acylation (17 — 18) and the deacylation (19 — 17) steps point to the involvement of a general base or nucleophile with a kinetically revealed piFCa value of ca. 7. A major difference here is that while the... 

Surprisingly few investigations appear to have been made into selective deacylation with the aid of enzymes. a-Chymotrypsin is very sluggish in hydrolyzing acetates of simple nucleosides and nucleotides, but dihydrocinnamic (3-phenylpropanoic) esters appear to be more satisfactory as substrates, and, with such derivatives of nucleosides, it seems that selective deacylation may be achieved enzymi-cally.178 Thus, enzymic hydrolysis of 2 -deoxy-3, 5 -di-0-(dihydrocin-namoyl)uridine gave the 3 -ester as the only intermediate to the... 

Figure 17.19 Rates of hydrolysis of two families of esters by a hydrolase, chymotrypsin. The esters of N-acetyl-L-phenylalanine exhibit very similar rates because the process in each case is limited by the same enzyme deacylation reaction (Zerner et al., 1964). The esters of N-benzoyl glycine exhibit rates varying by more than a factor of 3 because their hydrolyses are mostly limited by the initial enzyme acylation step (Epand and Wilson, 1963).

The Chymotrypsin Mechanism Involves Acylation and Deacylation of a Ser Residue... 

Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least 109. It does not catalyze a direct attack of water on the peptide bond instead, a transient covalent acyl-enzyme intermediate is formed. The reaction thus has two distinct phases. In the acylation phase, the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme. In the deacylation phase, the ester linkage is hydrolyzed and the nonacylated enzyme regenerated. 

FIGURE 6-19 Pre-steady state kinetic evidence for an acyl-enzyme intermediate. The hydrolysis of p-nitrophenylacetate by chymotrypsin is measured by release of p-nitrophenoi (a colored product). Initially, the reaction releases a rapid burst of p-nitrophenol nearly stoichiometric with the amount of enzyme present. This reflects the fast acylation phase of the reaction. The subsequent rate is slower, because enzyme turnover is limited by the rate of the slower deacylation phase. 

MECHANISM FIGURE 6-21 Hydrolytic cleavage of a peptide bond by chymotrypsin. The reaction has two phases. In the acylation phase (steps to ), formation of a covalent acyl-enzyme intermediate is coupled to cleavage of the peptide bond. In the deacylation phase (steps to ), deacylation regenerates the free enzyme this is essentially the reverse of the acylation phase, with water mirroring, in reverse, the role of the amine component of the substrate. Chymotrypsin Mechanism... 

Chromophoric acyl group,4,5 The spectrum of the furylacryloyl group depends on the polarity of the surrounding medium, and also on the nature of the moiety to which it is attached. The spectrum of furylacryloyl-L-tyrosine ethyl ester changes slightly when it is bound to chymotrypsin. There are also further changes on formation of the acylenzyme and on the subsequent hydrolysis. The rate constants for acylation and deacylation and the dissociation constant of the Michaelis complex may be measured by the appropriate experiments. 

There are also nonthematic methods that allow the formation of acylenzymes under conditions where they are stable, so that they can be stored in a syringe in a stopped-flow spectrophotometer. For example, it is possible to synthesize certain nonspecific acylenzymes and store them at low pH.9 12 When they are restored to high pH, they are found to deacylate at the rate expected from the steady state kinetics. This approach has been extended to cover specific acylenzymes. When acyl-L-tryptophan derivatives are incubated with chymotrypsin at pH 3 to 4, the acylenzyme accumulates. The solution may then be pH-jumped by mixing it with a concentrated high-pH buffer in the stopped-flow spectrophotometer.1314 The deacylation rate has been measured by the proflavin displacement method and by using furylacrylolyl compounds. 

Figure 16 4 The crystal structure of indolylacryloyl-chymotrypsin. [From R. Henderson, J. Molec. Biol. 54, 341 (1970).] Note that the carbonyl oxygen of this nonspecific acylenzyme is not bound between the NH groups of Ser-195 and Gly-193, but is nonproductively linked to His-57 by a hydrogen-bounded water molecule. This is the acylenzyme that was found to deacylate at the same rate in solution and in the crystal (Chapter 1). [G. L. Rossi and S. A. Bernhard, J. Molec. Biol. 49, 85 (1970).]...

A reaction looked at earlier simulates borate inhibition of serine proteinases.33 Resorufin acetate (234) is proposed as an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed when the more usual p-nitrophcnyl acetate is used as a substrate. The steady-state values are the same for the two substrates, which is expected if the slow deacylation step involves a common intermediate. Experiments show that the acetate can bind to chymotrypsin other than at the active site.210 Brownian dynamics simulations of the encounter kinetics between the active site of an acetylcholinesterase and a charged substrate together with ah initio quantum chemical calculations using the 3-21G set to probe the transformation of the Michaelis complex into a covalently bound tetrahedral intermediate have been carried out.211 The Glu 199 residue located near the enzyme active triad boosts acetylcholinesterase activity by increasing the encounter rate due to the favourable modification of the electric field inside the enzyme and by stabilization of the TS for the first chemical step of catalysis.211... 

Evidence for the tetrahedral intermediate includes a Hammett p constant of+2.1 for the deacylation reaction of substituted benzoyl-chymotrypsins and the formation of tetrahedral complexes with many inhibitors, such as boronates, sulfonyl fluorides, peptide aldehydes, and peptidyl trifluoromethyl ketones. In these last the chemical shift of the imidazole proton is 18.9 ppm, indicating a good low-barrier H-bond, and the pJQ of the imidazolium is 12.1, indicating that it is stabilized by 7.3 kcal mol 1 compared to substrate-free chymotrypsin. The imidazole in effect is a much stronger base, facilitating proton removal from the serine. 

This mechanism is similar to that of the serine protease a-chymotrypsin. In comparison to a-chymotrypsin, however, which features a general acid-base mechanism, the antibody requires a hydroxide ion (OH-) to initiate the deacylation step. The enzyme liberates both products P1 and P2 early in the catalytic cycle and very rapidly whereas the antibody liberates both products at the end of the catalytic cycle only. Even given all the similarities between enzymes and antibodies, one thus cannot expect complete analogy down to mechanistic details. However, the specificity of enzymes and antibodies can be very similar, as Table 18.1 corroborates. 

In addition, as is shown in Table 13, the deacylation which is represented in kd is increased by a factor of more than 103 in poly(PHA-IM-am), compared with poly(PHA-am). As is observed in the deacylation which is catalysed by imidazole moiety in the case of a-chymotrypsin, the catalysis by imidazole moiety in the deacylation of the acylated hydroxamic acid is considered. The turnover number, turnover hi poly(PHA-IM-am) is also high. The deacylation rate of poly(PHA-IM-am)... 

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