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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK032822-15
Application #
2749438
Study Section
Biochemistry Study Section (BIO)
Program Officer
Laughlin, Maren R
Project Start
1984-01-01
Project End
2000-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
15
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Buchbinder, J L; Rath, V L; Fletterick, R J (2001) Structural relationships among regulated and unregulated phosphorylases. Annu Rev Biophys Biomol Struct 30:191-209
Butte, M J; Hwang, P K; Mobley, W C et al. (1998) Crystal structure of neurotrophin-3 homodimer shows distinct regions are used to bind its receptors. Biochemistry 37:16846-52
Buchbinder, J L; Luong, C B; Browner, M F et al. (1997) Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation. Biochemistry 36:8039-44
Rath, V L; Lin, K; Hwang, P K et al. (1996) The evolution of an allosteric site in phosphorylase. Structure 4:463-73
Lin, K; Rath, V L; Dai, S C et al. (1996) A protein phosphorylation switch at the conserved allosteric site in GP. Science 273:1539-42
Buchbinder, J L; Fletterick, R J (1996) Role of the active site gate of glycogen phosphorylase in allosteric inhibition and substrate binding. J Biol Chem 271:22305-9
Crerar, M M; Karlsson, O; Fletterick, R J et al. (1995) Chimeric muscle and brain glycogen phosphorylases define protein domains governing isozyme-specific responses to allosteric activation. J Biol Chem 270:13748-56
Lin, K; Hwang, P K; Fletterick, R J (1995) Mechanism of regulation in yeast glycogen phosphorylase. J Biol Chem 270:26833-9
Buchbinder, J L; Guinovart, J J; Fletterick, R J (1995) Mutations in paired alpha-helices at the subunit interface of glycogen phosphorylase alter homotropic and heterotropic cooperativity. Biochemistry 34:6423-32
Rath, V L; Fletterick, R J (1994) Parallel evolution in two homologues of phosphorylase. Nat Struct Biol 1:681-90

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