Studies on possible phosphoryl enzyme formation in catalysis by hexokinase, pyruvate kinase, and glucose 6-phosphatase.

One possible mode of enzymic phosphoryli transfer involves formation of phosphoenzymes as intermediates. Thus far, however, convincing evidence for the formation of such intermediates has been obtained only for the phosphoglucomutase reaction (I, 2) and, to a lesser extent, for the phosphoglyceromutase reaction (3). Suggestive evidence has been reported for the participation of phosphoenzyme intermediates in certain phosphatase reactions. Participation of such intermediates was postulated by Hass and Byrne (4) and by Neuhaus and Byrne (5) for the glucose 6-phosphatase and phosphoserine phosphatase reactions, respectively. The findings of igren et al. (6-8) that radioactively labeled inorganic orthophosphate becomes attached to serine residues of alkaline phosphatase supports their contention that the catalysis involves phosphorylated enzyme intermediates. The catalysis of phosphoryl transfer by phosphatases (4, 5) is in accord with participation of phosphoenzyme intermediates, but may also be explained by alternate mechanisms (9). Isotope exchange studies have given evidence against formation of phosphoenzymes in kinase reactions, as shown by the lack of catalysis of exchange of pyruvate-Cl4 with phosphoenolpyruvate by pyruvate kinase (lo), of acetate-Cl4 with acetyl phosphate by acetate kinase (II), of glucose-U4 with glucose 6-phosphate by hexokinase (12)) and of radioactively labeled adenosine diphosphate with adenosine triphosphate by creatine kinase (13). Further, Callaghan and Weber (14) recently demonstrated that I?2 of the terminal phosphoryl group of ATP is not incorporated into rabbit muscle myokinase during catalysis. In contrast,