A Study of the Mechanism of “phosphotransferase” Activity by Use of Radioactive Phosphorus* by Bernard Axelrod

“Phosphotransferase” activity, which has been previously described (l), involves the enzymic transfer of phosphate from various aryl phosphates to particular hydroxyl compounds, apparently without the mediation of a nucleotide. It was indicated in this work that the phosphate group was transferred directly from the donor to the acceptor compound. The main basis for this belief was the observation at the enzyme plus inorganic phosphate did not transfer phosphate to the cceptor compound. Nevertheless it remained possible that the accepted phosphate arose from the pool of inorganic phosphate (which is always present since “phosphotransferase” activity is always accompanied by phosphstase activity), and the cleavage of the donor phosphate compound only provided the energy for the esterification. It has now been possible through the use of P3* to prove the direct transfer of phosphate from donor to acceptor. This has been shown in two ways. When unlabeled p-nitrophenyl phosphate was used as a phosphate donor in the presence of labeled inorganic phosphate, the synthesized phosphate ester contained virtually no labeled phosphate. But when the same donor contained labeled phosphate and was used with unlabeled inorganic phosphate, the synthesized ester did contain labeled phosphate as active as that present in the donor. In summary, the tests were conducted by subjecting p-nitrophenyl phosphate to the action of purified citrus phosphatase (which exhibits “phosphotransferase” activity very markedly) in the presence of methyl alcohol and inorganic phosphate. After incubation the increase in free nitrophenol was measured, showing the amount of nitrophenyl phosphate split. At the same time the increase in inorganic phosphate was measured to determine how much of the phosphate split from the substrate had been reesterified to methyl phosphate, as explained in the earlier paper (1). The methyl phosphate thus formed was isolated from the reaction mixture, and the specific radioactivity of the phosphorus was det,ermined. As this value is independent of the amount recovered, considerable loss in yields