Comparison of the Relative Rates of the Chymotrypsin-catalyzed and Nonenzymic Reactions of Esters with Hydroxylamine and Water.

The formation of an acyl-enzyme intermediate in the chymotrypsin-catalyzed reactions of esters (l-3) is widely held to be established, although some data do not quantitatively fit the theory (4, 5) and there is some dissent from this view. One of the simplest and most convincing ways of demonstrating the formation of an acyl-enzyme intermediate is to show that the chymotrypsin-catalyzed solvolysis, in the presence of hydroxylamine, of different esters containing the same acyl group produces precisely the same ratio of hydroxamic acid and carboxylic acid. This was shown for 11 esters of hippuric acid (2). This is, of course, a necessary condition for the acyl-enzyme theory. The conclusion that a common intermediate is involved rests upon the expectation that in reactions not involving a common intermediate, structural differences in the alcohol portions of the hippurate esters would result in different relative rates of reaction with water and hydroxylamine. This “selectivity” almost certainly can be predicted, since no constant ratio of two rates has ever been found for a series of reactions, except in cases in which a common intermediate is involved. This expected selectivity toward nucleophils is dramatically illustrated in the reactions of thiobutyl and p-nitrophenyl acetates with hydroxylamine (6, 7). The former compound reacts to produce predominantly acethydroxamic acid, while the latter forms predominantly 0-acetylhydroxylamine. In this paper the relative rates of reaction with hydroxylamine and hydroxide ion are measured for four of the esters used in our previous study. The ratios of these rates are compared with those of the enzymecatalyzed solvolysis. The argument for a common intermediate implies that if the structure of the acyl-enzyme is altered, the fraction of ester converted to hydroxamic acid will change. We have observed previously that different fractions were obtained with the use of OLand b-chymotrypsin. A comparison of data for furoyl, hippuryl, n-tyrosine, and N-acetyl-L-tyrosine esters (8, 2, 9) shows that wide variations in the structure of the acyl group