Flexibility in the Phosphorylase Catalytic Reaction

When rabbit muscle phosphorylase reconstituted with pyridoxal (5’)-diphospho(l)-a-~-glucose is incubated with glycogen, its glucosyl moiety is transferred to the nonreducing end of glycogen with the formation of a new a-1,4-glucosidic linkage. This finding provided the first evidence for the direct phosphate-phosphate interaction between the coenzyme pyridoxal 5’phosphate and the substrate a-D-glUCOSe 1-phosphate in the phosphorylase catalytic reaction (Takagi, M., Fukui, T., and Shimomura, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3716-3719). We have examined whether pyridoxal(5’)triphospho(l)-a-~-glucose can act in a similar manner to the diphospho compound or not. In the absence of glucan the enzyme-bound triphospho compound was stable for 1 day at pH 6-9. In the presence of glucan, however, its glucosidic linkage was cleaved, and the glucosyl moiety liberated was transferred to glycogen with the formation of a new a1,4-glucosidic linkage. Allosteric activator AMP accelerated the reaction and allosteric inhibitor glucose 6-phosphate showed the reverse effect. The pH optimum of the reaction was pH 8.1-8.4. Mg2+ slightly but significantly accelerated the reaction, whereas Mn2+ and Ca2+ inhibited the reaction. These results indicate that the glucosyltransfer from the triphospho compound occurs in an identical manner to that from the diphospho compound. Based on the present and previous data, we discuss the catalytic mechanism of phosphorylase, especially in comparison with that of phosphoryltransferases.