CHARACTERIZATION OF AN INHIBITORY METAL-BINDING SITE IN CARBOXYPEPTIDASE-A

The specificity of metal ion inhibition of bovine carboxypeptidase A ([CPD)Zn]) catalysis is examined under stopped-flow conditions with the use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent K(I) values of 2.4 X 10(-5), 4.8 X 10(-5), and 1.1 X 10(-2) M in 0.5 M NaCl at pH 7.5 and 25-degrees-C. The k(cat)/K(m) value, 7.3 X 10(6) M-1 s-1, is affected less than 10% at 1 X 10(-4) M Mn(II) or Cu(II) and at 1 X 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pK(LH) 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([CPD)Co]). The spectra of the transient [(CPD)Co].Zn and stable [(CPD)Co].Pb dimetallic enzyme species are both characterized by a maximum at 560 nm (DELTA-epsilon-560 = +60 M-1 cm-1) and shoulders at 520 and 585 nm. Spectral titrations of the [(CPD)Co].Pb complex with azide show that Pb(II) prevents azide binding to the catalytic Co(II) ion, providing further support for the idea that the inhibitory metal monohydroxide binds to the catalytic metal ion. The marked metal ion specificity of the inhibitory site can in part be accounted for by the stabilities of monohydroxide complexes of the metal ions examined. On the basis of charge considerations in the active site, the metal monohydroxide complex likely also carries one anion (Cl-) from the supporting electrolyte; the K(I) values for the neutral complexes Zn(OH)Cl and Pb(OH)Cl are approximately 5 x 10(-7) M. If the inhibitory metal monohydroxide complex is charge neutralized by a multidentate ligand, an even lower inhibition constant can be anticipated. Such inhibitory complexes could be very important to regulatory and/or toxicological processes of zinc enzymes.