Carboxypeptidase A: mechanism of zinc inhibition.
Zinc ions competitively inhibit carboxypeptidase A from bovine pancreas. The state(s) of hydroxylation of zinc and their possible site(s) of interaction with the enzyme have been investigated by determining the strength of zinc inhibition over pH range 4.6-10.5. The inhibition kinetics were recorded under stopped-flow conditions using the alpha-Val isozyme and the peptide substrate Dns-Gly-Ala-Phe in 0.5 M NaCl at 25 degrees C. The pH dependence of pKI follows a pattern which indicates that the enzyme is selectively inhibited by zinc monohydroxide, ZnOH+ (KI = 7.1 X 10(-7) M). The formation of the inhibitory ZnOH+ complex from fully hydrated Zn2+ is characterized by an ionization constant of 9.05, and the consecutive conversion of ZnOH+ to Zn(OH)2, Zn(OH)3-, and Zn(OH)4(2-) complexes takes place with ionization constants of 9.75, 10.1, and 10.5, respectively. Ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for binding of the ZnOH+ complex. The enzymatic activity (kcat/Km) is influenced by three ionizable groups: pKEH2 5.78, pKEH 8.60, and pKE 10.2. Since the values of pKLH and pKEH2 are virtually identical, it is possible that the inhibitory ZnOH+ complex interacts with the group responsible for pKEH2. Previous studies have suggested that pKEH2 reflects the ionization of Glu-270 and its interaction with a water molecule coordinated to the catalytic zinc ion. It is proposed that the inhibitory zinc ion binds to the carboxylate of Glu-270 and that the inhibition process is specific for zinc monohydroxide because it allows the formation of a stabilizing hydroxide bridge between the inhibitory and catalytic zinc ions.
Larsen
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KS
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts.
Auld
D S
DS
D000818:Animals; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D043422:Carboxypeptidases A; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects; D015032:Zinc / Q000494:pharmacology*
D007527:Isoenzymes; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc