Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b

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Abstract
The binding to [rabbit muscle] glycogen phosphorylase b of G-6-P and Pi (respectively, allosteric inhibitor and substrate/activator of the enzyme) were studied in the crytal at 0.3 nm (3 .ANG.) resolution. G-6-P binds in the .alpha.-configuration at a site that is close to the AMP allsoteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for G-6-P involves contacts to 2 cationic residues, Arg-309 and Lys-247. This site is also occupied in the Pi binding studies and is therfore 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 (2.7 .ANG.) away. The glucose moiety of G-6-P and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that G-6-P inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The transmission of allosteric conformational changes may involve an increase in affinity at phosphate-binding sites and relative movements of .alpha.-helices. In order to study G-6-P 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.