Abstract
Arg‐127 stabilizes the oxyanion of the tetrahedral intermediate formed during Zn2+ carboxypeptidase A‐catalyzed hydrolysis. Mutant carboxypeptidases lacking Arg‐127 exhibit substantially reduced rates of hydrolysis with the change manifest almost entirely in kcat (kcat/Km is decreased by 104 for R127A). Therefore, Arg‐127 stabilizes the enzyme‐transition state complex but not the ground state enzyme‐substrate complex (Phillips, M.A., Fletterick, R., & Rutter, W.J., 1990, J. Biol. Chem. 265, 20692–20698). The addition of guanidine, methylguanidine, or ethylguanidine to R127A increases the kcat for hydrolysis of Bz‐gly‐(o)phe by 102 without changing the Km. Dissociation constants (Kd) for the guanidine derivatives range from 0.1 to 0.5 M. The binding affinity for the transition state analog Cbz‐phe‐alap(o)ala is increased similarly by 102; in contrast, the binding affinity of the ground state inhibitor benzylsuccinic acid is not altered. Thus, guanidine derivatives mimic Arg‐127 in stabilizing the rate‐limiting transition state. Hydrolysis of Bz‐gly‐(o)phe by wild‐type carboxypeptidase, R127K, or R127M is not substantially affected by guanidine derivatives. Additionally, primary amines do not change the activity of R127A. These observations imply that guanidine binds in the cavity vacated by Arg‐127 specifically and in a productive conformation for catalysis.