Direct phosphate-phosphate interaction between coenzyme and substrate in the catalytic reaction of glycogen phosphorylase

Glycogen phosphorylase contains firmly bound pyridoxal 5′-phosphate (PLP), and catalyzes the reversible transfer of a glucosyl moiety between glucose-1-phosphate (G-1-P) and α-1,4-glucan. X-ray crystallographic studies revealed that PLP is located in a pocket where the phosphate group of PLP is pointed toward the G-1-P binding site. We have synthesized pyridoxal(5′)diphospho(1)-α-d-glucose, as a model compound for the phosphate-phosphate interaction between PLP and G-1-P, and reconstituted the enzyme with this compound. The resulting enzyme is catalytically inactive in itself, but, in the presence of glucan, the glycosyl moiety of this compound is transferred to the glucan forming a new α-1,4-glucosidic linkage along with the production of pyridoxal 5′-diphosphate. This glucosyltransfer is similar to the normal catalytic reaction in various aspects, although the rate is smaller in the order of three. AMP accelerates the transfer about 24 times compared with the reaction in its absence. We have more recently used pyridoxal(5′)triphospho(1)-α-D-glucose to reconstitute the enzyme. In the presence of glucan, the compound bound to enzyme is gradually degraded to pyridoxal 5′-triphosphate. This reaction is essentially dependent on AMP, and proceeds several times more slowly than the glucosyltransfer from the diphospho compound. These results provide evidence for the direct phosphate-phosphate interaction between the coenzyme and the substrate in the normal enzyme reaction, and seem to reflect a rather wide allowance in regard to this interaction.