| In an attempt to demonstrate that the side chain residue of Tyr-248 of the common pancreatic metalloenzyme carboxypeptidase A is involved in the catalytic process of amide scission, hydrolysis of CbzGlyGlyPhe by nitro(Tyr-248)carboxypeptidase A over the pH range 4.88-8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K(,m) is scarcely affected. The peptidase activity has a bell-shaped pH dependency for k(,cat)/K(,m) characterized by the following parameters: pK(,E1) of 6.37 (+OR-) 0.19 and pK(,E2) of 6.60 (+OR-) 0.17, but only a single apparent pK of 5.59 (+OR-) 0.06 in k(,cat). A spectroscopic pK of 6.75 (+OR-) 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK(,E2) in the k(,cat)/K(,m) profile, which appears to be shifted from a higher value, pK(,E2) of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k(,cat) profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, wherein ionization of the metal-bound water molecule (pK of 6.37) prevents substrate binding and the phenolate of Tyr-248 (pK of 9 for native enzyme, and 6.6 for nitroenzyme) functions as a general base, is described with support from inhibition studies, chemical modification, and crystallographic evidence for the enzyme.;As part of mechanistic investigation on the role of the active site metal ion, studies of cleavage of a thiopeptide by Zn- and Cd-carboxypeptidase A have been carried out. Substitution of the active site zinc ion of carboxypeptidase A by cadmium yields an enzyme inactive towards ordinary peptide substrates. However, a substrate analog (Bz-GlyNHCH(,2)CSPheOH) containing a thioamide linkage at the scissile position is cleaved to the thioacid. The kinetic parameters in comparison with hydrolysis of ordinary carboxamides by native carboxypeptidase are not greatly changed; k(,cat) is decreased 100-fold and K(,m) is increased by approximately 10. The pH dependency (k(,cat), k(,cat)/K(,m)) has exactly the same form. The thiopeptide is much less efficiently cleaved by native (zinc) caboxypeptidase A. This cadmium-sulfur synergism suports a mechanism wherein the substrate amide is activated by metal ion coordination to its (thio)carbonyl. |
