Excess zinc ions are a competitive inhibitor for carboxypeptidase A.
The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
Hirose
J
J
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Ando
S
S
Kidani
Y
Y
D000818:Animals; D001667:Binding, Competitive; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D043422:Carboxypeptidases A; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D010649:Phenylalanine; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology*
D010649:Phenylalanine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc