Studies on the Allosteric Activation of Glycogen Phosphorylase b by Nucleotides

The allosteric mechanism of phosphorylase b activation by inosine monophosphate has been investigated and compared with AMP activation of this enzyme.Enzyme activity was measured in the direction of glycogen synthesis.In contrast to AMP, IMP affected the V,,, but not the enzyme affinity toward glucose-l-P.The plots of initial rate (vi) against glucose-l-P concentration for IMP activation were sigmoidal, having Hill coefficients of 2, the homotropic cooperativity of glucose-l-P being independent of nucleotide concentration.The plots of vi against IMP concentration were sigmoidal at low glucose-l-P concentrations but hyperbolic at high substrate.In the presence of 0.3 M sodium fluoride the kinetics was normalized, resulting in an increased nucleotide aflinity.Under these conditions the K, values of AMP and IMP activation were both independent of substrate concentration; only the V,,, changed with increasing glucose-l-P.The kinetic data suggest that the mechanism of nucleotide activation results in a two-stage allosteric transition of the enzyme, viz., a change in both catalytic efficiency and enzyme affinity toward glucose-l-P.This proposed mechanism receives support from the following physical criteria.AMP, IMP, and glucose-1 -P show varying degrees of protection against cold inactivation and the removal of pyridoxal phosphate from the enzyme.While AMP causes partial dimerization of phosphorylase b, IMP may dimerize the enzyme only in the presence of glucose-l-P.Muscle glycogen phosphorylase b (cr-1,4-glucan :orthophosphate glucosyltransferase, EC 2.4.