THE BINDING OF 2-DEOXY-D-GLUCOSE 6-PHOSPHATE TO GLYCOGEN-PHOSPHORYLASE-B - KINETIC AND CRYSTALLOGRAPHIC STUDIES

Kinetic and crystallographic studies have characterized the effect of 2-deoxy-glucose 6-phosphate on the catalytic and structural properties of glycogen phosphorylase b. Previous work on the binding of glucose 6-phosphate, a potent physiological inhibitor of the enzyme, to T stat e phosphorylase b in the crystal showed that the inhibitor binds at th e allosteric site and induces substantial conformational changes that affect the subunit-subunit interface. The hydrogen-bond from the O-2 h ydroxyl of glucose 6-phosphate to the main-chain oxygen of Val40' repr esents the only hydrogen bond from the sugar to the other subunit, and this interaction appears important for promoting a more ''tensed'' st ructure than native T state phosphorylase b. 2-Deoxy-glucose 6-phospha te acts competitively with both the activator AMP and the substrate gl ucose 1-phosphate, with K-i values of 0.53 mM and 1.23 mM, respectivel y The binding of 2-deoxy-glucose 6-phosphate to T state glycogen phosp horylase b in the crystal, has been investigated and the complex phosp horylase b: 2-deoxy-glucose 6-phosphate has been refined to give cryst allographic R factor of 17.3%, for data between 8 Angstrom and 2.3 Ang strom. 2-Deoxy-glucose 6-phosphate binds at the allosteric site as the a anomer and adopts a different conformation compared to glucose 6-ph osphate. The two conformations differ by 160 degrees in the torsion an gle about the C-5-C-6 bend. The contacts from the phosphate group are essentially identical to those made by the phosphate of glucose 6-phos phate but the 2-deoxy glucosyl moiety binds in a quite different orien tation compared to the glucosyl of glucose 6-phosphate. 2-Deoxy-glucos e 6-phosphate can be accommodated in the allosteric site with very lit tle change in the protein, while structural comparisons show that the phosphorylase b: 2-deoxy-glucose g-phosphate complex structure is over all more similar to a glucose-like complex than to the Glc-6-P complex structure. (C) 1995 Academic Press Limited