Heterotropic Interactions of Ligands with Phosphorylase b

Abstract

The interaction of rabbit muscle glycogen phosphorylase b (EC 2.4.1.1) with pairs of ligands was examined. The ESR spectrum of a spin label, covalently attached to the protein, provided information about dissociation constants, formation of ternary complexes and both negative and positive interactions between different ligand pairs. AMP competes with a series of nucleotides (ADP, ATP, CMP and cytosine), but with adenosine a ternary enzyme.cntdot.AMP.cntdot.adenosine complex can be formed. ADP binding is tight and ADP inhibits the AMP activation of phosphorylase b in a physiologically important concentration range. The substrates glucose 1-phosphate and glycogen tighten AMP binding in the ternary complex as does the competitive inhibitor UDPG. Inorganic phosphate is different in this respect. Gluconolactone, a transition state analogue, competes with glucose 1-phosphate (but not with glycogen) but does not prevent completely the binding of the sugar phosphate. The effect of glucose 6-phosphate on phosphorylase is rather complex as it formally competes with both AMP and UDPG probably mediated by a conformational change and not by direct interactions with these 2 ligands. Glycerol 2-phosphate, a commonly used buffer for phosphorylase, also shows complex interactions.