Arginine 127 stabilizes the transition state in carboxypeptidase.
Crystallographic studies suggest that Arg-127 is a key amino acid in the hydrolysis of peptides and esters by carboxypeptidase A. The guanidinium group of Arg-127 is hypothesized to stabilize the oxyanion of the tetrahedral intermediate formed by the attack of water on the scissile carbonyl bond. We have replaced this amino acid in rat carboxypeptidase A1 with lysine (R127K), methionine (R127M), and alanine (R127A), in order to define the role of Arg-127 in carboxypeptidase catalyzed hydrolysis. The wild-type and mutant enzymes were expressed in yeast and purified. Kinetic studies show that Arg-127 substitution decreases kcat for both ester and amide substrates, whereas Km is relatively unchanged; for R127M and R127A this corresponds to a 6 kcal/mol decrease in transition state stabilization of the rate-limiting step. The binding affinity for the phosphonate transition state analog, Cbz-Phe-Ala(P)-OAla, was decreased by 5.4 kcal/mol, whereas binding affinity for the ground state inhibitor, DL-benzylsuccinic acid, was decreased by only 1.7 kcal/mol for R127M. Electrostatic calculations employing a finite difference solution to the Poisson-Boltzmann equation predict that the positive charge of Arg-127 should stabilize the transition state by 6-8 kcal/mol. Therefore, the experimental and theoretical data suggest that the primary role of Arg-127 is stabilization of the transition state through electrostatic interaction with the oxyanion.
Phillips
M A
MA
Hormone Research Institute, University of California, San Francisco 94143-0534.
Fletterick
R
R
Rutter
W J
WJ
D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D043422:Carboxypeptidases A; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005822:Genetic Vectors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011199:Potentiometry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D015345:Oligonucleotide Probes; D001120:Arginine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A