Inositol hexaphosphate and its Cu(II) coordinate complex as inhibitors of intestinal alkaline phosphatase

Myo-Inositol hexaphosphate (phytic acid), a naturally occurring plant constituent capable of forming coordinate complexes with polyvalent cations, reversibly inhibits the hydrolysis of p-nitrophenyl phosphate by alkaline phosphatase. Kinetics of the inhibition is of the strictly competitive type with Ki equal to 260 microM at pH 8.0 and 25 degrees. A similar result is also obtained in the presence of an inositol hexaphosphate-cupric ion coordinate complex. This inhibition of substrate hydrolysis by either inositol hexaphosphate or its Cu(II) complex is to be differentiated from the effect of the latter on alkaline phosphatase in the absence of substrate. In this case total inhibition of the enzyme is obtained in a time-dependent process which apparently involves a metal ion exchange reaction, i.e., substitution of the enzyme's zinc atoms for copper atoms. The effect of inositol hexaphosphate on alkaline phosphatase contrasts with that of inorganic phosphate which gives inhibition kinetics of the partially competitive type with Ki (for its effect on KM) 17.1 microM; a value close to that of KM for the substrate (15.2 microM). At a low phosphate concentration relative to that of inositol hexaphosphate in the assay solution the observed inhibition was partially additive. At a higher phosphate to inositol hexaphosphate ratio, the inhibition was essentially a phosphate effect. This provides additional support for the conclusion that Inositol hexaphosphate and, by extension, an inositol hexaphosphate-Cu(II) complex inhibits alkaline phosphatase by interaction at the active sites in competition with the substrate.