Metal binding to yeast aminopeptidase I.

Binding of Zn(II) and Co(II) to homogeneous dodecameric aminopeptidase I from yeast was studied. Apparent binding constants were estimated from the dependence of enzyme activity on metal levels in solution as established by metal buffer systems. The binding curves were sigmoidal with Hill coefficients of 1.2-1.6, depending on the metal and on pH. Metal concentrations at half-saturation were 28 nM with Zn(II), and 390 nM with Co(II) at pH 7.0, they increased with decreasing pH. Equilibrium binding studies yielded binding stoichiometries of 0.5-0.6 mol metal/mol subunit for both Zn(II) and Co(II). However, after oxidation of Co(II)-substituted enzyme with H2O2 almost stoichiometric amounts of cobalt (greater than 0.9 mol/mol subunit) were found. The oxidized enzyme was inactive and did not exchange cobalt with the solvent. Native aminopeptidase I was also affected by H2O2, however only after prolonged incubation and at a much lower rate. Apoenzyme modified by ethoxyformic anhydride could not be reconstituted by Zn(II) or Co(II). On the other hand, either metal afforded full protection against ethoxyformic anhydride. This finding, and the pH dependence of stability constants suggest that histidine residues are involved in metal binding. Both the reconstitution of active enzyme from apoprotein and metal and its inactivation due to loss of metal are slow processes with half-times in the minute range. The rate of reconstitution did not depend on Zn(II) concentration. The rates of metal loss were not enhanced by chelating agents containing carboxylate functions. In contrast, chelators coordinating via nitrogen atoms and 2,3-dimercaptopropanol accelerated dissociation. A model is proposed that accounts for the substoichiometric metal contents of aminopeptidase I and for the slow time course of reconstitution. It is assumed that reconstitution involves a slow, monomolecular transition, leading to an active enzyme conformation with lower affinity for metal ions.