The phosphorylated intermediate in the phosphoglyceromutase reaction

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Abstract
High-voltage paper-electrophoresis methods have been used for the separation of 32P-labeled phosphoesters. Evidence is presented which indicates that 32P-labeled phosphopeptides, obtained after acid hydrolysis of phosphoglyceromutase incubated with impure 2, 3-di[32P]phosphoglycerate, are derived from phosphoglucomutase contamination. The hydrolysis of 2, 3-di[32P] phosphoglycerate by phosphoglyceromutase has been studied. After an apparent instantaneous hydrolysis of 1 mole of coenzyme/mole of enzyme the reaction proceeds at a very low rate. This hydrolysis seems to be due to the destruction of an enzyme-coenzyme complex. The proportions of the decomposition products of the complex vary according to further handling (pH of ionophoresis). The inorganic [32P]-phosphate produced by the hydrolysis of the complex and the inorganic [32P] phosphate produced by the slow phosphatase activity can be differentiated by the ability of the former to be incorporated into non-radioactive substrate before enzyme denaturation. The effect of enzyme concentration on the stoichiometry of the slow phosphatase hydrolysis of the diphosphoglycerate is described and this suggests that it may occur via 2 independent reactions, 1 of them being the decomposition of the enzyme-coenzyme complex on standing.