Hippuryl-alpha-methylphenylalanine and hippuryl-alpha-methylphenyllactic acid as substrates for carboxypeptidase A. Syntheses, kinetic evaluation and mechanistic implication.

(R)- and (S)-Hippuryl-alpha-methylphenylalanine [(R)- and (S)-Hipp-alpha-MePhe] and (S)-hippuryl-alpha-methylphenyllactic acid [(S)-Hipp-alpha-MeOPhe] were synthesized and evaluated as substrates for carboxypeptidase A (CPA) in an effort to shed further light on the catalytic mechanism of the enzyme. The rate of CPA-catalyzed hydrolysis of (S)-Hipp-alpha-MePhe was reduced by 105-fold compared with that of (S)-Hipp-Phe, but the hydrolysis rate of (S)-Hipp-OPhe was lowered by only 6.8-fold by the introduction of a methyl group at the alpha-position. (R)-Hipp-alpha-MePhe failed to be hydrolyzed initially, then started to undergo hydrolysis in about 2 h at a much reduced rate. The results of present study may be envisioned on the basis of the proposition that while peptide substrate is hydrolyzed via a tetrahedral transition state formed by the attack of the zinc-bound water molecule at the peptide carbonyl carbon, ester hydrolysis takes the path that involves an anhydride intermediate generated by the attack of the carboxylate of Glu-270 at the ester carbonyl carbon.