The Role of Pyridoxal Phosphate in the Catalysis of Glycogen Phosphorylases

Glycogen phosphorylases catalyze the degradation of glycogen by phosphate (or arsenate) to glucose 1‐phosphate (or glucose + arsenate). All glycogen phosphorylases that have been studied so far contain pyridoxal 5′‐phosphate, a vitamin B 6 ‐derivative, as cofactor. Removal of the cofactor results in an inactive apoenzyme. However, reduction of the azomethine bond linking pyridoxal phosphate to an ϵ‐aminolysyl side chain of the enzyme with NaBH 4 does not inactivate glycogen phosphorylase. If therefore the cofactor should be involved in catalysis in glycogen phosphorylase it must function differently from all other classical pyridoxal phosphate dependent enzymes, for these are inactivated by reduction. 31 P‐NMR spectroscopy has revealed that the 5′‐phosphate group of pyridoxal phosphate is present in catalytically active forms of glycogen phosphorylases as dianion in a hydrophobic environment shielded from aqueous solvent. Covalent and/or allosteric activation of muscle glycogen phosphorylases is accompanied by a transition of the monoprotonated form to the dianionic form of the phosphate group of the cofactor. We now report on such ionization changes in unregulated active potato‐ and E. coli maltodextrin phosphorylases on binding of glucose and oligosaccharides and following catalytic turnover, i.e. arsenolysis of α‐1,4‐glycosidic bonds. (Like glycogen phosphorylases, maltodextrin phosphorylases belong to the class of α‐glucan phosphorylases.) The results of experiments carried out by our group together with recent findings on the three dimensional structure of crystalline muscle glycogen phosphorylases indicate a participation of the dianionic phosphate group as proton acceptor for the glucosyl transfer to and from the glucosyl acceptor. Although other interpretations are not excluded, at present little doubt remains that in the case of glycogen phosphorylases the dianionic phosphate group of the cofactor functions in catalysis.