A hypothesis that the variation in the apparent pAf for deacylation of substituted acyl-chymotrypsins is associated with a preequilibrium hydration of the acyl-enzyme to form a tetrahedral intermediate has been tested. The crux of this test is based upon the fact that in the aminolysis of an acyl-enzyme the tetrahedral intermediate is different from that formed in the hydrolysis reaction so that (a) the equilibrium for formation of a tetrahedral intermediate may differ with an amine nucleophile as compared with water; (b) the rate-determining step in the reaction of an amine may involve tetrahedral intermediate formation rather than breakdown; and (c) the pK of the enzyme histidine residue responsible for activity may vary when a tetrahedral intermediate contains an amine rather than a water molecule. Any of these factors can be expected to result in a difference in the pH dependence for aminolysis as compared with hydrolysis if the hypothesis under review is correct. The apparent pK's for hydrolysis of benzoyl-, p-nitrobenzoyl-, /j-methoxybenzoyl-, 3,5-dinitrobenzoyl-, and furoyl-chymotrypsin are 7. 36, 7.22, 7.32, 6.95, and 7.10, respectively. The p/Ts for reaction of methoxylamine with benzoyl-and furoyl-chymotrypsin are 7.41 and 7.14, respectively. From this it is concluded that the pK variations observed for hydrolysis are not associated with the accumulation of a tetrahedral intermediate which has a substantially different pK than that of the acylenzyme.
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