Comparison of AMP and NADH binding to glycogen phosphorylase b.
The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D003461:Crystallography; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D046911:Macromolecular Substances; D008968:Molecular Conformation; D009243:NAD / Q000378:metabolism*; D010763:Phosphorylase b / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits
D046911:Macromolecular Substances; D009243:NAD; D000249:Adenosine Monophosphate; D010763:Phosphorylase b; D006005:Phosphorylases