The long term goal of this proposal is to understand the molecular mechanisms involved in regulating glycogen metabolism. Glycogen metabolism is regulated by the change in activity of glycogen phosphorylase; the enzyme modulates its catalytic activity through the binding of effector molecules and by reversible covalent phosphorylation. Phosphorylase in humans exists as a family of three isozymes, brain, liver, and muscle, that are markedly diverged in sequence and regulatory components of phosphorylases determine their specialized activity and physiological roles. The major objectives include: (1) understanding detailed mechanisms of phosphoregulation, with emphasis on the phosphopeptide element-where and how it positions, how this sequence is protected or made accessible to the kinase, and what changes occur in its conformation and functional role; (2) examining and comparing tertiary & quaternary states of differing functional modes of glycogen phosphorylase, as influenced by substrates and allosteric effectors; and (3) understanding the design principles of the phosphorylase regulatory mechanism. As a model for addressing these objectives, yeast phosphorylase shares core structural attributes of human phosphorylases and may offer a scaffold for completing the regulatory architecture found in human isozymes. Hence, the research design comprises characterizing the structure and function of yeast phosphorylase using X-ray crystallography, enzyme kinetic, and biochemical analysis. Structural solutions are proposed for several forms of phosphorylated as well as unphosphorylated yeast phosphorylase. Crystallographic analysis will be complemented with kinetic and biophysical analysis of activation, inhibition, and phosphorylation control in native and mutated enzyme forms. Crystal structures and biochemical data for a variety of enzyme complexes will determine what structural changes accompany activation or inhibition in yeasty phosphorylase. These changes will be compared to what has been observed in the mammalian muscle enzyme in order to synthesize a stronger understanding of the regulatory mechanisms in the human phosphorylase isozymes. These basic mechanisms have strong eventual implications for developing therapy to help persons with diabetes and other metabolic disorders, such as hepatic glycogen storage diseases.
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