45864-G4
Chemical Mechanism of a Unique, Cofactor-Independent Decarboxylase
The initial focus of this grant was to fund our explorations of the mechanism of PadC, a cofactor independent decarboxylase involved in lignin degradation. In the past year, the focus of this project has switched toward a different enzyme, namely human glucokinase. Human glucokinase catalyzes the ATP-dependent phosphorylation of glucose in the first step of glycolysis. This chemical transformation is the rate-limiting step of glucose metabolism in the human liver and pancreas and glucokinase is generally considered to be the body's principal regulator of glucose homeostasis. The mechanism of human glucokinase is interesting because this monomeric enzyme is allosterically regulated by its substrate, D-glucose. The steady-state velocity of glucose phosphorylation is not hyperbolic, but instead displays a sigmoidal response to increasing glucose concentrations. The goal of our research program is to elucidate the structural and mechanistic basis for kinetic cooperativity in human glucokinase. Within the last year, we have achieved solid progress on understanding the regulation of human glucokinase regulation activity. We have optimized a bacterial expression system that affords the production of large quantities of recombinant enzyme. We have also used a genetic selection strategy to identify eleven new activating mutations in the human glucokinase gene. Currently, we are conducting steady-state kinetic assays on these variants to determine the kinetic constants, kcat, Km, and K0.5. We are also employing a number of biophysical techniques, including transient state kinetics, fluorescence spectroscopy, X-ray crystallography and analytical ultracentrifugation to test the hypothesis that each activated variant functions to enhance catalysis by favoring a more compact enzyme state. Our most notable discovery was the identification of I211F, a variant enzyme that represents the most hyperactive glucokinase enzyme identified to date. We speculate that this lesions causes a fatal form of hyperinsulinemia of infancy.
