Protein Science, Vol 1, Issue 4 517-521, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

Guanidine derivatives restore activity to carboxypeptidase lacking arginine-127

M. A. PHILLIPS, L. HEDSTROM and W. J. RUTTER
Hormone Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143-0534

Arg-127 stabilizes the oxyanion of the tetrahedral intermediate formed during Zn(2+) carboxypeptidase A-catalyzed hydrolysis. Mutant carboxypeptidases lacking Arg-127 exhibit substantially reduced rates of hydrolysis with the change manifest almost entirely in k(cat) (k(cat)/K(m) is decreased by 10(4) 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 k(cat) for hydrolysis of Bz-gly-(o)phe by 10(2) without changing the K(m). Dissociation constants (K(d)) for the guanidine derivatives range from 0.1 to 0.5 M. The binding affinity for the transition state analog Cbz-phe-ala(P)(o)ala is increased similarly by 10(2); 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.
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