Carboxypeptidase A-Catalyzed Hydrolysis of α-(Acylamino)cinnamoyl Derivatives of L-β-Phenyllactate and L-Phenylalaninate: Evidence for Acyl-Enzyme Intermediates

Article abstract of DOI:10.1021/ja00301a025

The (CPA) carboxypeptidase A-catalyzed hydrolysis of α-(acetylamino)- (1), α-(benzoylamino)- (2), or α-<(2-naphthoyl)amino>cinnamoyl ester (3) of L-β-phenyllactate manifested small values of both k_cat and K_mapp.On the other hand, the corresponding amides of L-Phe showed enhanced k_cat and unaffected K_mapp values.At -2 deg C, accumulation of an intermediate was observed spectrophotometrically immediately after mixing of 6x10^-5 M CPA with 4x10^-5 M 3.This is the most stable (in terms of half-life and K_mapp) intermediate ever reported for the CPA-catalyzed reactions.For 2 and 3, k_cat was independent of pH over pH 5.5-9.5.Although attempts to trap the intermediate with external or intramolecular trapping reagents were unseccesful, the very small K_mapp and the pH independence of k_cat for 2 and 3 provide evidence that shows that the accumulating intermediate is the anhydride acyl-CPA intermediate.The temperature dependence and the D2O effect were measured for the k_cat values of 2 and 3.The different effects of the α-(acylamino)cynnamoyl groups on the kinetic parameters for esters and for peptides were explained in terms of a single mechanism with the intermediacy of an acyl-enzyme.