Understanding allosteric control is a central question in biology. Glycogen phosphorylase degrades glycogen to release energy in a form that the cell can use immediately. In humans, there are three distinct forms of the enzyme named after the tissues in which they are preferentially expressed; liver, muscle and brain phosphorylase. Liver phosphorylase regulates blood sugar by sensing glucose levels and providing glucose as needed. The muscle enzyme provides energy for movement. The third isozyme is more stringently regulated to respond to intracellular metabolism in brain tissue. Phosphorylases are activated or inhibited according to intracellular metabolite concentrations and by extracellular hormonal signals. The principal metabolite effectors of activity in vivo are glucose, AMP, ATP, Glc-6-P and some unidentified molecule that is a purine analog. Hormones, including glucocorticoids and epinepherine, control the activity of phosphorylase through the reciprocal action of kinases and phosphatases. Phosphorylation of the enzyme occurs at a single site, Ser 14. Both phosphorylation and the binding of effectors alter the catalytic activity of the enzyme by changing the conformation of the protein. Many of these conformational changes have been defined by X-ray crystallography experiments. This proposal describes molecular, biochemical and kinetic experiments to further determine the specific amino acids and secondary structural elements of phosphorylase that are responsible for the activation and inhibition of the enzyme activity. Variants of human phosphorylase isozymes will be produced using recombinant DNA methods and site-directed mutagenesis. The variants will be specifically designed to test current and new models of the structural basis of allosteric control in phosphorylase. The functional properties will be characterized by biochemical and kinetic analysis. The three-dimensional structures and kinetic behavior of these enzymes will be correlated. The primary goal is to determine the structural basis underlying the differential regulation of the three human phosphorylase enzymes observed in vivo. The proposed project seeks to gain new understanding about the protein components required for the functioning of complex, tailored allosteric mechanisms which are exquisitely expressed in phosphorylase and basic to the understanding of how structure dictates function. An understanding of these basic mechanisms could potentially be used to design drugs that regulate blood glucose levels. This knowledge can be used to understand how allosteric mechanisms regulate the activity of other important proteins. It is widely appreciated that regulation by phosphorylation plays a critical role in many important biological processes such as signal transduction and cell cycle control, to name but two examples.
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