Inorganic Pyrophosphate-Glucose Phosphotransferase Activity Associated with Alkaline Phosphatase of Escherichia coli

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Abstract Highly purified commercial alkaline phosphatase preparations from Escherichia coli have been shown to catalyze the transfer of a phosphoryl group of inorganic pyrophosphate to the hydroxyl group attached to carbon atom 6 of glucose. Phosphotransferase activity also was detected with relatively crude chicken intestinal alkaline phosphatase preparations. The E. coli enzyme was further characterized relative to catalytic properties. Adenosine 5'-phosphate, diphosphate, and triphosphate, cytidine 5'-phosphate and diphosphate, guanosine triphosphate, and mannose 6-phosphate functioned as alternate phosphoryl donors. Activity with PPi as substrate was observed between pH 5 and 9, with a maximum at pH 7.9. Km (PPi), 0.83 mm, was independent of glucose concentration. Maximal activity was observed with 1.25 m glucose; higher concentrations of this hexose inhibited both phosphotransferase and phosphohydrolase activities of the enzyme. Maximally, in the presence of 1.25 m glucose, the phosphotransferase activity involved the participation of 9% of reacting inorganic pyrophosphate; the remaining 91% of the reacted phosphoanhydride substrate was hydrolyzed. The phosphotransferase activity resembled inorganic pyrophosphatase activity of the preparation with respect to effects of pH, pyrophosphate concentration requirement, and thermolability at 30° in the absence of substrates. Glucose 6-phosphatase and AMPase activities of the preparation also were progressively inactivated by mild heating in a manner similar to that of phosphotransferase and inorganic pyrophosphatase activities; however, β-glycerol phosphate hydrolysis was more stable, and p-nitrophenyl phosphate hydrolysis activity was considerably more labile to this treatment than were the other four activities studied. A number of alternative explanations for differences in stability of these hydrolytic activities attributed to alkaline phosphatase are presented. While the high glucose requirement and relatively low ratio of phosphotransferase to phosphohydrolase activity of the enzyme make a biologically significant role for the synthetic activity questionable, it is suggested that the phosphotransferase reaction may provide a tool for further studies relating to the reaction mechanism of alkaline phosphatase.