Kinetic Study on the Acylation Step of α-Chymotrypsin-Catalyzed Hydrolysis of Acylimidazole

A Model Reaction of Specific Peptide Substrate Activated by Binding to the Enzyme

Abstract

As a model reaction of the enzyme-bound activated peptide substrates in proteinase-catalyzed hydrolysis, the α-chymotrypsin-catalyzed hydrolysis of three acylimidazoles (trans-cinnamoyl-imidazole, indoleacryloylimidazole, and furylacryloylimidazole) was studied, especially as regards the acylation process. The complicated pH-dependences of the reactions, due mainly to the existence of the protonated forms of these acylimidazoles, were analyzed based on a reaction scheme considering the so-called inactive monomeric enzyme and the dimeric enzyme. The intrinsic reaction parameters for individual species were evaluated.
The acylation through the mono-protonated enzyme-substrate complex (ESH) reflects the rate of attack by Ser-195 O^γ on the carbonyl carbon of acylimidazolium, where the formation of the possible tetrahedral intermediate is rate-determining. The acylation via the non-protonated enzyme-substrate complex (ES) is intermediate between those of esters (as regards the single bond character of the cleaved linkage) and of amides (with respect to the proton transfer as a key process of the acylation). This was confirmed by the observation of acceleration of the acylation rate upon addition of external proton donors. Based on these results, the mechanism of acylation in peptide substrate hydrolysis is discussed.