Interaction of zinc ions with arsanilazotyrosine-248 carboxypeptidase A

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Abstract
The interaction between arsanilazotyrosine-248 carboxypeptidase A [(Azo-CPD)Zn] and excess Zn2+ was studied by stopped-flow and spectrophotometric methods at pH 8.2 and 7.7, I = 0.5 M (NaCl), and 25.degree. C. When excess Zn2+ bind to arsanilazotyrosine-248 carboxypeptidase A, the characteristic red color, which arises from the intramolecular complex of the arsanilazotyrosine-248 residue with the active site Zn of the enzyme, changes to yellow with the inhibition of peptidase activity of the enzyme. Excess Zn2+ have 2 binding sites for arsanilazotyrosine-248 carboxypeptidase A, and the binding constants of the 1st site (3.9 .RTM. 105 M-1 at pH 8.2; 7.1 .RTM. 104 M-1 at pH 7.7) are much larger than those of the 2nd site (1.8 .RTM. 103 M-1 at pH 8.2; 7 .RTM. 102 M-1 at pH 7.7). The binding of excess Zn2+ to the 1st site is completely correlated with the inhibition of the enzyme peptidase activity and the color change of the enzyme. The results can be understood in terms of Zn2+ reacting with only 1 of 3 conformational states of arsanilazotyrosine-248 carboxypeptidase A [Harrison, L. W., Auld, D. S. and Vallee, B. L. (1975)]. The 2nd-order rate constants for the binding of excess Zn2+ to arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn] + Zn2+ .fwdarw. [(Azo-CPD)-Zn] .cntdot. Zn2+) are 4.3 .RTM. 106 and 8.4 .RTM. 105 M-1 s-1, at pH 8.2 and 7.7, respectively, and the 1st order rate constants for the dissociation of Zn2+ from the adduct between Zn2+ and arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn] .cntdot. Zn2+ .fwdarw. [(Azo-CPD)Zn] + Zn2+) are 11 and 12 s-1, respectively. Excess Zn2+ apparently promoted the inhibition of the peptidase activity and the color change from red to yellow through binding specificially to 1 conformational state of arsanilazotyrosine-248 carboxypeptidase A.