Studies on allosteric phenomena in glycogen phosphorylaseb

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Abstract
This article attempts to trace, from a personal point of view, the history of discoveries of allosteric phenomena in phosphorylaseb and the later development of systematic attempts to fit the data into comprehensive theoretical models. Work from our own laboratory is emphasized, but we try to integrate this into the results from other investigators and show their contributions to our ideas and experiments. Finally, some recent unpublished data is presented together with some conclusions and predictions from a new hypothesis. The discoveries byCarl andGerty Cori of the activation of phosphorylase by AMP, the inhibition by glucose and the enzymatic interconversion of two forms of the enzyme with different control properties helped lay the foundations of our present understanding of allosteric mechanisms. The later discovery of the oligomeric nature of phosphorylase and its relationship to AMP binding served as a basis for many years of research into the structurefunction relationships of phosphorylase and other enzymes. Data showing that AMP lowers the entropy of activation is discussed with respect to the role of the nucleotide and its binding close to the active site. The discovery of the control of phosphorylaseb by common metabolites and the impetus this gave to the intensive kinetic studies of the last ten years, wherein fitting to theoretical models has been a common feature, is reviewed. Chemical and physical probes were sought in order to find evidence for the conformational states and transitions predicted by kinetics. Allosteric inhibitors and activators antagonized each other with respect to rates of isocynate inhibition while substrate provided additional protection to enzyme saturated with AMP, suggesting an additional conformational state. This was confirmed by effects on sulfhydryl group reactivity, and it was also shown that substrate by itself increased the reactivity of a particular —SH group. Thus AMP by itself causes the basic T state to assume the R′ conformational state, substrate induces this into an R state, but substrate by itself promotes what we may term an S state. Other laboratories have also presented kinetic and physico-chemical evidence for the R′, R and S states. Further evidence for the conformational states mentioned above was obtained by examining the effect of ligands on the quenching of the pyridoxal phosphate (PLP) cofactor by iodide or on the quantum yield of the PLP. The allosteric inhibitor, ATP, increased both the accessibility of PLP to iodide and the quantum yield, suggesting induction of a different conformational state which we term I, evidence for which has also been presented by other groups using other probes. Surprisingly, however, ATP or glucose-6-P, which antagonize substrate binding in the presence of AMP, show positive homotropic cooperativity with substrate in the absence of AMP. Thus the inhibitor and the substrate improve each others' binding, as indicated by the PLP fluorescence, and this suggests that both may bind simultaneously and tightly to a conformational state which we term T′. These suggestions are incorporated into a model and the implications for the structurefunction relationships in the enzyme are discussed.

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