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.
Lee
M
M
Center for Biofunctional Molecules and Department of Chemistry, Pohang University of Science and Technology, South Korea.
Kim
D H
DH
D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D005069:Evaluation Studies as Topic; D006626:Hippurates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007773:Lactates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D006626:Hippurates; D007773:Lactates; C411085:hippuryl-methylphenylalanine; C121218:hippurylphenyllactic acid; D010649:Phenylalanine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A