pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15)

AbstractThimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clarifies the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pKa of 5.7 and a basic limb with pKa of 8.8. The acidic limb can be attributed to protonation of a residue affecting kcat while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin