Abstract
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.
Highlights
Peptides can bind to the apoenzyme, whereas esters cannot [7], and it has been hypothesized that a binding mode involving Arg-127 exists for peptides but not esters. These results suggest that Arg-127 will have a more important function in the hydrolysis of peptides than for esters( 1)
Much is known about CPA, the mechanism of hydrolysis is still not well understood
Hydrolysis is believed to proceed by attack of Zr?+-bound water on the peptide bond to form a tetrahedral intermediate, but the exact nature and function of the amino acids involved is still unclear
Summary
CPA substrates, and CPA inhibitors were purchased from Sigma with the exceptions of CLCPL, which had been prepared previously [8], carbonapthoxyphenylalanine, which was purchased from Pfaltz and Bauer, Inc., Waterbury, CT, and Cbz-Phe-Ala(P)-. OAla, which was a generous gift from fir. The gene encodinp rat proCPA [9] was cloned into Bluescript (Strata&me). DNA was isolated from Esckrichia coli strain RZ1032 (Hfr KL 16 PUl45 flvs A/61-62)ldut. Zbd-279::T&d, supE44) harbo&g the CPA plasmidyas recommended by Stratagene. Mutagenesis was performed by the method of Kunkel(l0).
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