title: Characterization of an inhibitory metal binding site in carboxypeptidase A.
abstract: The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with 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 KI 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 kcat/Km 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 (pKLH 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]).(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: K S
authors.initials: KS
authors.lastname: Larsen
authors.forename: D S
authors.initials: DS
authors.lastname: Auld
mesh_terms: D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000494:pharmacology*; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides; D007700:Kinetics; D007854:Lead / Q000494:pharmacology*; D010179:Pancreas / Q000201:enzymology; D013053:Spectrophotometry; D015032:Zinc / Q000494:pharmacology*
chemical_list: D006878:Hydroxides; D002104:Cadmium; D007854:Lead; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc