A bicarbonate ion as a general base in the mechanism of peptide hydrolysisby dizinc leucine aminopeptidase

The active sites of aminopeptidase A (PepA) from Escherichia coli and leuci ne aminopeptidase from bovine lens are isostructural, as shown by x-ray str uctures at 2.5 Angstrom and 1.6 Angstrom resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zin c ions. It is shown that PepA is activated about 10-fold by bicarbonate whe n L-leucine p-nitroanilide is used as a substrate. No activation by bicarbo nate ions is found for mutants R356A, R356K, R356M, and R356E of PepA, In t he suggested mechanism, the bicarbonate anion is proposed to facilitate pro ton transfer from a zinc bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is simil ar to the catalytic role of carboxylate side chains in the presumed mechani sms of other dizinc or monozinc peptidases, A mutational analysis shows tha t Arg-356 influences activity by binding the bicarbonate ion but is not ess ential for activity. Mutation of the catalytic Lys-282 reduces k(cat)/K-m a bout 10,000-fold.