Inactivation of intestinal alkaline phosphatase by inositol hexaphosphate-Cu (II) coordinate complexes

Alkaline phosphatase (APase) was greater than 99% inactivated upon incubation with myo-inositol hexakisphosphate (IHP) and Cu(II) ions. In the absence of Cu(II), IHP did not inactivate the enzyme. Likewise, cupric ions alone did not produce inactivation. Reactions of APase with IHP plus Cu(II) were competitively inhibited by zinc ions. In contrast to the marked effect of (IHP-Cu) chelate complexes on APase activity, the complexes of IHP with either Zn(II) or Mn(II) had no discernable effect. Both the extent and the rate of activity loss were dependent on the combined IHP and Cu(II) concentration. At an IHP to Cu(II) ratio of 11.6, the extent of inactivation was approximately proportional to the Cu(II) concentration with maximal inactivation attained above 10 microM. Under the same conditions, a nonlinear relation (saturation kinetics) was observed between the pseudo first-order rate constants for the reaction and the IHP and Cu(II) concentration. On the basis of adherence of the data to a mechanism involving an intermediate whose concentration was rate determining, it was suggested that a ternary complexes composed of the apoprotein, the catalytic site zinc ions, and one or more specific IHP-Cu(II) complex ( IHP-Cu]*) may be the first step along the reaction coordinate. Relevant to this possibility which assumes active site interaction is the fact that both IHP alone and (IHP-Cu) complexes are good competitive inhibitors of p-nitrophenyl phosphate hydrolysis under the same solution conditions wherein APase inactivation occurs in the absence of substrate. Rates of enzyme inactivation are decreased with an increase in pH from 6.5 to 8.0. They are also dependent upon buffer type and concentration, apparently related to their association constants for cupric ion binding. Over and above such specific effects, rates of inactivation are also reduced with an increase in ionic strength. Depending on the ratio and concentrations of IHP and Cu(II) used in the reaction with APase, subsequent exposure to EDTA followed by assay in the presence of Zn(II) gave recoveries of activity ranging from 60% to 100%. Both the prior inactivated enzyme (containing IHP and cupric ions) in the presence of EDTA and the native APase upon simultaneous exposure to IHP, Cu(II), and EDTA were slowly and irreversibly inactivated. Correction for this effect gave reconstitution of activity of the (IHP-Cu)-inactivated APase by Zn(II) addition equivalent to that which could be obtained by EDTA-treatment of the native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)