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 RSPO 3 Na 2 (R = —CH 2 CH 2 NH 2 , —CH 2 CH 2 NHCOCH 3 , —CH 2 COO, or —CH 2 CH 2 COOC 2 H 5 ) 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 MgCl 2 . The K m and V max values obtained, as well as the amount of MgCl 2 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 ROPO 2 SKH (R = —CH 3 , —CH 2 CH 3 , or [See PDF for structure]NO 2 ) were completely resistant to hydrolysis by alkaline phosphatases from Escherichia coli and from intestine. The O -substituted monoesters of thiophosphoric acid (10 -7 m ) inhibited the enzymic hydrolysis of both cysteamine S -phosphate and p -nitrophenyl phosphate (10 -3 m ) at a 10-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.