The phosphorylated intermediate in the phosphoglyceromutase reaction.

1. High-voltage paper-electrophoresis methods have been used for the separation of (32)P-labelled phosphoesters. 2. Evidence is presented which indicates that (32)P-labelled phosphopeptides, obtained after acid hydrolysis of phosphoglyceromutase incubated with impure 2,3-di[(32)P]phosphoglycerate, are derived from phosphoglucomutase contamination. 3. The hydrolysis of 2,3-di[(32)P]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. 4. 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). 5. The inorganic [(32)P]phosphate produced by the hydrolysis of the complex and the inorganic [(32)P]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. 6. The effect of enzyme concentration on the stoicheiometry of the slow phosphatase hydrolysis of the diphosphoglycerate is described and this suggests that it may occur via two independent reactions, one of them being the decomposition of the enzyme-coenzyme complex on standing.