It is proposed to continue and expand the crystallographic and structural analysis of glycogen phosphorylase at high resolution and to elucidate the three-dimensional structure of its several activated (R-state) conformational states. Phosphorylase is a complex (97.4 K dalton) allosteric enzyme regulated by a number of physiologically important ligands and by phosphorylation/dephosphorylation under neural and hormonal control. Phosphorylase plays a critical role in muscle and liver tissue in the mobilization and homeostasis of glucose. At least two disease states (Hers and McArdle's disease) can be traced to mutations which affect the structure and regulation of phosphorylase. This research addresses the mechanism of allosteric regulation of phosphorylase by effectors and covalent introconversion, the characterization of its multiple conformational states, and the mechanism by which conformation changes are communicated homotropically and heterotropically among effector loci and the active sites within the catalytic dimer. The ultimate goal is to contribute to the development of structural models for complex, allosterically regulated enzyme systems. The structural data will also be used to develop and test models for the catalytic mechanism for phophorylase, the most abundant (though mechanistically unique) of Pyridozal containing enzymes. Specific goals are as follows: 1) Determination of an accurate, highly refined atomic model for the glucose inhibited (T-state) conformation of glycogen phophorylase with 1.9 Angstrom data measured with the multi-wire Detector facility in the laboratory of Prof. Xuong at U.C. San Diego. 2) Determination of the structures at 2.5-3.0 Angstrom resolution of effector activated conformational states obtained by diffusion of ligands into phophorylase crystals. Complexes to be studied are: maltopentaose + orthophosphate, maltopentaose + selenate (in which oligosaccharide is bound at the catalytic site), maltopentoase + orthophosphate + AMP. 3) Solution of the structure of pyridoxal pyrophosphoryl phosphorylase, a covalent analog of the binary enzyme-orthophosphate substrate complex which is crystallized in the R state and diffracts to 3.0 Angstrom resolution. 4) Detailed comparison, in collaboration with Prof. L. Johnson of Oxford, of the high resolution structures of phosphorylase a and b to elucidate the conformational effects of the a P b conversion.
