Kinetics of the carboxypeptidase A-catalyzed hydrolysis of α-(benzoylamino)cinnamoyl derivatives of various amino acids

Kinetics of the carboxypeptidase A-catalyzed hydrolysis of α-(benzoylamino)cinnamoyl derivatives of various amino acids were measured. The C-terminal amino acid residue of the amide substrates were phenylalanine (Phe), p-substituted phenylalanines (p-OH, p-NO₂, p-Cl, p-I, p-CH₃p-C₂H₅, and p-CH(CH₃)₂), tryptophan (Trp), and phenylglycine(phenylGly). Introduction of the p-OH, p-Cl, p-NO₂, or p-CH₃ group to the leaving Phe did not lead to considerable changes in the reactivity, while that of the p-I or p-C₂H₅ group decreased the reactivity remarkably and that of the p-CH(CH₃)₂ group abolished the reactivity. Substitution of Trp for the leaving Phe did not alter the reactivity appreciably, whereas that of phenylGly for the leaving Phe reduced the reactivity to a large degree. The seemingly scattered pattern of the structure-reactivity relationship was explained by assuming that the hydrophobic pocket in the active site of the enzyme has limited height, which is comparable to the thickness of the benzene ring.