Mechanism of the phosphorylase reaction

α‐Glucan phosphorylases from rabbit skeletal muscle, potato tubers and Escherichia coli catalyze the utilization of 2,6‐anhydro‐1‐deoxy‐ d ‐ gluco ‐hept‐1‐enitol (heptenitol) in the presence of arsenate or phosphate. 1 H‐NMR analysis in the presence of 2 H 2 O and arsenate indicated formation of 1‐[1‐ 2 H]deoxy‐α‐ d ‐glucoheptulose with rates comparable to the arsenolysis of poly‐ or oligosaccharides. The reaction depends on the presence of a dianionic 5′‐phosphate group of pyridoxal in the active conformation of the phosphorylases. Heptenitol is the first known substrate of α‐glucan phosphorylases which does not require a primer. This is explained by the finding that heptenitol is exclusively used as substrate for the degradative pathway of the phosphorylase reaction where it competes with polysaccharide substrates. In the presence of phosphate the reaction product is 1‐deoxy‐α‐ d ‐ gluco ‐heptulose 2‐phosphate (heptulose‐2‐ P ), which subsequently inhibits the reaction. This characterizes heptulose‐2‐ P as an enzyme‐derived inhibitor. The K i = 1.9 × 10 −6 M with potato phosphorylase suggests the formation of a transition‐state‐like enzyme‐ligand complex. These findings, together with the fact that the phosphates of heptulose‐2‐ P and pyridoxal 5′‐phosphate are linked by hydrogen bridges [Klein, H. W., Im, M. J., Palm, D. & Helmreich, E. J. M. (1984) Biochemistry 23 , 5853–5861], make it likely that both phosphates are involved in phosphorylase catalysis. A catalytic mechanism of phosphorylase action is proposed in which a ‘mobile’ phosphate anion plays a versatile role. It serves as proton carrier for the substrate activation, it stabilizes the intermediate and acts as a nucleophile which can accept a glycosyl residue reversibly.