Catalytic reaction of glycogen phosphorylase reconstituted with a coenzyme-substrate conjugate

The role of pyridoxal 5'-phosphate in the catalytic mechanism of glycogen phosphorylase (EC 2.4.1.1) remains unresolved despite extensive investigation. A previous report from this laboratory (Takagi, M., Fukui, T., and Shimomura, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3716-3719) provided evidence for the direct interaction between the two phosphate groups of the coenzyme and a substrate alpha-D-glucose 1-phosphate. When apophosphorylase is reconstituted with pyridoxal (5')-diphospho(1)-alpha-D-glucose, the enzyme can transfer the glucose moiety to glycogen just as in the normal catalysis. We have studied the kinetics of the glucosyltransfer from this compound to glycogen. The normal and mimic reactions were similar in their kinetic parameters for glycogen and AMP and their activation energies. AMP and other nucleotides that activate the normal reaction could activate the mimic reaction as well. The log k/pH plot for the mimic reaction gave a bell-shaped curve with pKa = 6.90 and pKb = 8.84 at 25 degrees C. The apparent heats of ionization of the corresponding groups having the pKa and pKb were 6.9 and 3.0 kcal/mol, respectively. Reversibility of the glucosyltransfer from the coenzyme-substrate conjugate to glycogen could not be demonstrated, possibly because the equilibrium of this reaction lies further to the polysaccharide synthesis. Based on these and other data which confirm the role of the phosphate group of the coenzyme as an electrophile, we discuss the catalytic mechanism of glycogen phosphorylase. It is suggested that the imidazoyl group of His-376 acts as a nucleophile attacking the anomeric carbon of the substrate glucose 1-phosphate.