Glycogen phosphorylase is a highly regulated enzyme which catalyzes the breakdown of glycogen, a storage carbohydrate to glucose-1-phosphate, which can be readily metabolized. The processes by which glycogen is used and synthesized are critical to the correct functioning of tissues and numerous metabolic disorders are known to originate in the glycogen storage system. Phosphyrlase is perhaps the most complex allosteric enzyme yet characterized, responding to an array of metabolic nervous stimuli. Allosteric regulation is mediated through covalent modification, the binding of effector molecules and intersubunit cooperativity. Having determined the crystal structure of rabbit muscle phosphyrlase a at high resolution, this laboratory is in a unique position to investigate the structural problem of regulation. Preliminary experiments reveal that it is possible to generate a spectrum of increasingly activated conformations by introducing effectors into the crystals of phosphrylase. Structural changes are complex, linking effector sites with the catalytic site, and are not interpretable in terms of a simple model. We propose to define these structures at 3.0 Angrstom resolution or better, by combining sensitive data collection techiques with well-tested model building and refinement procedures. The structures of substrate activated forms will assist in elucidating the mechanism of allostery in phosphorylase and in defining the role of the covalently bound pyridoxal phosphate coenzyme essential to catalysis. Hepatic phosphorylase must respond to varying blucose fluxes in the maintenance of glucose hemeostasis. Accordingly, they respond differently to metabolically induced effectors. Because the phosphorylase structure is highly conserved we expect that the differing physiological functions arise from subtle changes at the effector sites. We propose to analyze the liver structure by diffraction techniques. Crystals of liver Pa in the presence of activators have been obtained in the laboratory, and studies are currently under way.