Solvent effects on the deacylation of acyl-chymotrypsins: a critical comment on the charge-relay hypothesis

Abstract

The pH dependencies for deacylation of a series of acyl-chymotrypsins, prepared by using peptide-free enzyme, are reported. The pKa′ values exhibit considerable variation, from 7.63 for α-benzamido-trans-cinnamoyl-chymotrypsin to 6.6 for 3,5-dinitrobenzoyl-chymotrypsin. It is proposed that the variation results from an interaction of variable strength between the carboxylate ion of Asp-102 and protonated His-57 and, further, that the kinetically determined values of the pKa′ are attributable in all cases to the ionization of His-57. High values for the pKa′ controlling deacylation are therefore indicative of a strong electrostatic interaction and low values reflect a relatively weak interaction. The effect of 20.4% (w/w) dioxane on the pKa′' associated with the deacylation of a numher of acyl-chymotrypsins is reported. The measured shifts in the value of the pKa′ provide strong evidence for this description of the Asp-His pair in acyl-chymotrypsins. In the hydrolysis of the specific substrate N-acetyl-L-tryptophan ethyl ester, the ionizing group controlling deacylation behaves as a simple cationic acid. The results clearly indicate that a charge relay is not involved in the hydrolysis of specific substrates and argue against a kinetically important role for aspartic acid acting as a base in the mechanism of action of the serine proteinases.