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The pH dependence for acylation of alpha-chymotrypsin by N-acetyltryptophan p-nitrophenyl-, p-nitrothiophenyl-, ethyl-, and thiolethyl esters has been studied by the stopped-flow technique. Values for the acylation rate constant, k2, and the binding constant, KS, were obtained by using measurements of phenolate release, for the p-nitrophenyl esters, and proflavin displacement, for the ethyl esters. The oxygen esters tested have slightly higher k2 values, and substantially higher KS values relative to the analogous thiol esters. Whereas k2/KS for the thiolethyl ester is higher than that for the analogous oxygen ester, the k2/KS values for oxy- and thio-p-nitrophenyl esters are nearly identical. These data are interpreted to indicate rate-determining formation of a tetrahedral intermediate in acylation of alpha-chymotrypsin by p-nitrophenyl esters, and rate-determining breakdown of such an intermediate in the case of the ethyl esters. It is also concluded that the oxygen to sulfur substitution causes a substantial increase in the proportion of nonproductive binding in these substrates. pH dependent k2 and KS values were used to calculate values for k1 and k-1, the binding and debinding rate constants for the two p-nitrophenyl compounds. This is the first such calculation based on experimentally determined acylation rate constants.

Dimeric alpha-chymotrypsin (D) formed over a range of low pH (3.7-5.2) showed a different specificity from monomeric alpha-chymotrypsin (M) for the acylation step, though the catalytic system functions as well in the dimeric enzyme as in the monomeric enzyme. For aliphatic substituted phenyl esters a bulky substituent at the ortho-position of the phenyl ring reduced the acylation rate of the dimeric enzyme compared with the monomeric enzyme. Especially for aliphatic p-nitrophenyl esters, the dissociation constants of the DS complexes were always smaller than those of the MS complexes, independent of the aliphatic chain length of the substrate. The intrinsic acylation rate constant of the dimeric enzyme increased with the chain length of the substrate. These catalytic properties are discussed with reference to the structures of the catalytic and binding sites of the dimeric enzyme.