Various physiological stimuli, e.g., hormones such as insulin and epinephrine, neural excitation and pharmacological agents influence gl ' vcogen metabolism in a variety of tissues by altering a cascade of enzymatic phosphorylation-dephosphorylation reactions. Although much is known about the sequence of reactions that occur in response to many biological signals, excepting insulin, little is known about the molecular aspects of these reactions. Our main aim is to provide basic information about mechanisms of specific phosphorylation-dephosphorylation reactions. What structural and chemical features do the enzymes recognize in their protein substrates? How does catalysis occur and how is it regulated? From our studies on specificity and mechanism, we anticipate both specific and general information will be derived leading to a better understanding of phosphorylation-dephosphorylation reactions. A rationale may be reached for the design of specific inhibitors. Such inhibitors may find use in studies of the action of insulin, other hormones, and physiological stimuli. The grant consists of two parts: (1) a study will be made of how the gamma catalytic subunit of phosphorylase kinase acts and how it is regulated, A bacterial expression system will be utilized to provide pure enzyme (normal and mutated forms) for various enzymatic, chemical and physical studies. The mechanism by which calcium activates the enzyme, the involvement of base catalysis in the reaction, and features influencing specificity will be examined; (2) an investigation will be made of mechanistic aspects of tyrosyl phosphorylation catalyzed by the insulin receptor kinase and dephosphorylation reactions by calcineurin and tyrosyl phosphatases. The influence of the chemical characteristics of the phenolic group in substrates on enzymatic reactions will be evaluated and a study will be made of the mechanistic relatedness of kinases and phosphatases.
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