ENZYMATIC MECHANISMS OF SULFUR NUCLEOSIDE METABOLISM

Institute For Cancer Research, Philadelphia PA

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Abstract

DESCRIPTION (adapted from applicant's abstract) S-adenosylmethionine (AdoMet) is the primary biological alkylating agent and occupies an essential role in the metabolism of all cells. Thus, enzymes involved in AdoMet dependent processes are targets for development of chemotherapeutic agents. The objectives of this research are to elucidate the active site structures and catalytic mechanisms of two of these enzymes. Studies of AdoMet synthetase (ATP:L-methionine S-adenosyltransferase) will elucidate the structural basis for inhibition by a newly found high affinity, slow binding inhibitor, the intermecliate analog diimidotriphosphate (O3P-NH-PO2-NH-PO3). Kinetic and spectroscopic studies will reveal the steps in formation of the enzyme-inhibitor complex, and the structure of the bound inhibitor. The crystal structure of AdoMet synthetase shows a flexible loop which gates access to the active site, an important catalytic event. The influence of the length and composition of the loop sequence on catalytic function will be unveiled. The dynamics of the loop will be characterized by EPR spectroscopy of a spin-labeled loop residue; whether substrates or products alter the loop dynamics will be elucidated. AdoMet synthetases from Archaea have very different sequences from those of the eukarya and prokarya, suggesting an altered catalytic strategy; the Methanococcus jannaschii synthetase will be characterized. Predicted differences in catalytic properties will be evaluated using structural and functional studies of the wild type enzyme and selected mutants. AdoMet decarboxylase catalyzes the reaction that directs the product to polyamine biosynthesis. The enzyme contains an unusual covalently attached pyruvate group that forms a Schiff base with the substrate as a reaction intermediate. The mechanisms of the enzyme from E. coli, which requires a divalent metal ion for activity and the metal independent enzyme from M. jannaschii will be elucidated. The rates and equilibria of the steps in the mechanism will be determined by presteady state kinetic methods. The active site structure in the free enzyme and complexes will be characterized by NMR of l3C-pyruvate enriched enzyme, and 13C and l5N enriched substrate. Magnetic resonance studies of Mn2+ complexes will reveal whether the divalent metal ion activator binds at the active site, perhaps coordinating the pyruvate to facilitate the Schiff base formation. The mechanism of inhibition by the chemotherapeutic agent methylglyoxal bis(guanylhydrazone) will be determined by kinetic and NMR measurements, revealing whether inhibition results from formation of a covalent adduct with the pyruvyl moiety.