Hydrolysis of picolinylprolines by prolidase. A general mechanism for the dual-metal ion containing aminopeptidases

Abstract

The velocity of enzymic cleavage of 4-substituted picolinylprolines by swine kidney prolidase approaches that of physiological dipeptides, but depends substantially upon the nature of the pyridine-ring substituent. The pH dependence of kcat/Km for picolinylproline is sigmoidal, with optimum activity on the acidic limb and a delimiting enzymic pKa of 6.6, unlike glycylproline (bell-shaped pH profile, maximum at pH 7.7). Productive chelation to an active site metal ion by the N terminus of substrates is indicated, with a water molecule ligated to that hyper(Lewis)acidic center prior to substrate binding supplying the pKa of 6.6. The rate-governing catalytic step differs according to the 4-substituent on the picolinyl residue; productive binding is slow in the case of electron-withdrawing groups, but subsequent nucleophilic addition to the metal ion-activated scissile linkage becomes controlling with more basic pyridine rings. Rate constants yield a Brønsted-type correlation with substrate pKa, providing a gauge of active-site Lewis acidity. A mechanism is suggested involving the cooperative participation of two especially acidic metal ions positioned adjacently within the active site (situated as in an homologous and structurally characterized aminopeptidase), with both serving to stabilize a bridging carboxamide-hydrate intermediate.