MECHANISM OF INOSINE MONOPHOSPHATE DEHYDROGENASE

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Inosine-5'-monophosphate dehydrogenase (IMP:NAD) oxidoreductase; IMPDH) catalyzes the rate limiting reaction in the biosynthesis of guanine nucleotides. IMPDH is an established target of anti-tumor and anti-viral chemotherapeutic agents. The goals of the proposed research are to determine the active site structure and catalytic mechanism of the form of IMPDH present in human tumors. The enzyme will be isolated from Escherichia coli which express large quantities of the enzyme from the cloned gene. An integrated kinetic, spectroscopic and affinity labelling approach will be employed in these studies. The steps in the IMPDH-catalyzed reaction pathway and the rate constants for those steps will be determined by a combination of steady state and presteady state kinetic studies and primary kinetic isotope effect determinations. These studies will reveal the order of substrate binding and product release, and the extent to which hydrogen transfer is rate limiting in catalysis. The chemical mechanism of catalysis will be determined by characterization of the structures of substrates, products and intermediate(s) bound at the active site. 13C NMR and 15N NMR of appropriately enriched substrates and products will be used in conjunction with ultraviolet spectroscopic studies. The purine ring tautomers and ionization states of IMP and the product XMP bound to the enzyme will be elucidated. The conformations of bound substrates will be determined from 1H NMR transferred nuclear Overhauser effects. These spectroscopic studies will provide a detailed characterization of the structures of the enzyme-bound forms of substrates, products and potentially also of intermediates. The mechanism of activation by monovalent cations, such as K+, will be determined. Kinetic studies will elucidate the effects of cation binding on catalytic efficiency and the selectivity of the enzyme for cations of various ionic radii. Equilibrium binding studies will reveal the stoichiometry of cation binding and NMR studies will reveal whether the ion binds at the active site or is an allosteric activator. Amino acids which contribute to the active site will be identified by affinity labelling using reactive IMP analogs and photoaffinity derivatives of IMP and NAD. How site-directed mutations of these residues affect substrate binding and catalytic efficiency will be determined. The results of these studies will form the basis for understanding the interaction of IMPDH with chemotherapeutically important compounds, and for the design of new inhibitors.