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 enzyme s of the chymotrypsin and subtilisin families. Our kinetic study using the thiono substrate, benzyloxycarbonyl-Gly-Pro[CS-NH]-2-naphthylamid e suggests that the putative oxyanion binding site is important in pro lyl oligopeptidase catalysis, although to a lesser extent than in the chymotrypsin-and subtilisin-catalyzed reactions. By using another thio no substrate, benzyloxycarbonyl-Gly[CS-NH]Pro-2-naphthylamide, it is d emonstrated that the distant S2P2 hydrogen bond (formed between the S2 subsite and P2 peptide residue) makes a greater contribution to catal ysis than does stabilization by the oxyanion binding site involved dir ectly in the bond cleavage. In contrast to the reactions catalyzed by chymotrypsin and subtilisin, no kinetic deuterium isotope effect is ap parent in the acylation of prolyl oligopeptidase measured either with the specific benzyloxycarbonyl-Gly-Pro-2-naphthylamide, or with the ve ry poor substrate, benzyloxycarbonyl-Gly-Pro[CS-NH]-2-naphthylamide. T his indicates that the rate-limiting conformational change is induced by the substrate.