Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b.
The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.
D000249:Adenosine Monophosphate; D000494:Allosteric Regulation; D000495:Allosteric Site; D001665:Binding Sites; D003461:Crystallography; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates; D007096:Imidoesters; D008958:Models, Molecular; D010710:Phosphates; D010763:Phosphorylase b; D006005:Phosphorylases; D011487:Protein Conformation
D005958:Glucosephosphates; D007096:Imidoesters; D010710:Phosphates; D000249:Adenosine Monophosphate; C007475:dimethylmalonimidate; D019298:Glucose-6-Phosphate; D010763:Phosphorylase b; D006005:Phosphorylases