Substrate selectivity in the action of alkaline and acid phosphatases.

Alkaline phosphatase of intestinal origin hydrolyzed the S-substituted monoesters of phosphorothioic acid of the type RSP03Na2 (R = -CH2CH2NH2, -CH,CH,NHCOCH,, -CH,COO, or -CH2CHsCOOCzHs) at the S-P bond to yield orthophosphate and the corresponding thioalcohols. The rate of enzymic hydrolysis of cysteamine S-phosphate was measured at pH 9.0 in 0.1 N sodium barbital buffer at different concentrations of substrate and MgC12. The K, and V,,, values obtained, as well as the amount of MgC12 required for complete activation of the enzyme, were similar to the corresponding values obtained when p-nitrophenyl phosphate was used as the substrate. In marked contrast, however, O-substituted monoesters of phosphorothioic acid of the type ROPOeSKH (R = -CH3, -CH&H,, or -aNOe) were completely resistant to hydrolysis by alkaline phosphatases from Escherichia coli and from intestine. The O-substituted monoesters of thiophosphoric acid (10M7 M) inhibited the enzymic hydrolysis of both cysteamine S-phosphate and p-nitrophenyl phosphate (10e3 M) at a lo-fold concentration of the enzyme. Acid phosphatases from wheat germ, potato, and prostate gland did not hydrolyze S-substituted monoesters of phosphorothioic acid at detectable rates, but did hydrolyze O-substituted monoesters of phosphorothioic acid at rates comparable with those obtained when p-nitrophenyl phosphate served as the substrate for these enzymes. These findings suggest that acid and alkaline phosphatases act by two different mechanisms.