Prolyl oligopeptidase catalysis. Reactions with thiono substrates reveal substrate-induced conformational change to be the rate-limiting step.

Prolyl oligopeptidase, a member of the new family of serine proteases, exhibits significant mechanistic differences compared with the enzymes of the chymotrypsin and subtilisin families. Our kinetic study using the thiono substrate, benzyloxycarbonyl-Gly-Pro[CS-NH]-2-naphthylamide suggests that the putative oxyanion binding site is important in prolyl oligopeptidase catalysis, although to a lesser extent than in the chymotrypsin- and subtilisin-catalyzed reactions. By using another thiono substrate, benzyloxycarbonyl-Gly[CS-NH]Pro-2-naphthylamide, it is demonstrated that the distant S2P2 hydrogen bond (formed between the S2 subsite and P2 peptide residue) makes a greater contribution to catalysis than does stabilization by the oxyanion binding site involved directly in the bond cleavage. In contrast to the reactions catalyzed by chymotrypsin and subtilisin, no kinetic deuterium isotope effect is apparent in the acylation of prolyl oligopeptidase measured either with the specific benzyloxycarbonyl-Gly-Pro-2-naphthylamide, or with the very poor substrate, benzyloxycarbonyl-Gly-Pro[CS-NH]-2-naphthylamide. This indicates that the rate-limiting conformational change is induced by the substrate.