The sulfur-containing adenine nucleoside derivatives S-adenosylmethionine (AdoMet) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) play essential roles in the metabolism of all cells. The objectives of this research are to elucidate the active site structures and catalytic mechanisms of two enzymes which catalyze the biosynthesis of these important metabolites. These enzymes are S-adenosylmethionine synthetase (ATP:L-methionine-S- adenosyltransferase) and adenosine-phosphosulfate kinase (ATP:adenosine- 5'-phosphosulfate-3'-phosphotransferase). Studies of AdoMet synthetase will use site-directed mutagenesis, followed by in-depth characterization of the mutant enzymes, to reveal the roles of active site amino acid residues in the two reactions that the enzyme catalyzes: 1) formation of AdoMet and tripolyphosphate from ATP and methionine, and 2) the hydrolysis of the tripolyphosphate to pyrophosphate and orthophosphate. Site-directed mutagenesis of known active site residues is now possible since the X-ray crystal structure has been solved for the Escherichia coli AdoMet synthetase which is cloned and overproduced. Residues potentially involved in catalysis of C-O bond cleavage during AdoMet formation and in activation of the gamma-phosphoryl group for hydrolysis are targeted. AdoMet synthetase requires two freely dissociable divalent cations per active site for catalytic activity. The residues that provide ligands to these metal ions will be determined by a combination of site-directed mutagenesis and EPR spectroscopic methods with Mn2+ and VO2+ as probes. The changes in metal ligation that occur during the reaction cycle will be determined by rapid freeze quenching of enzyme samples during the course of a single turnover, followed by analysis using Electron Paramagnetic Resonance methods. The adenosine-5'-phosphosulfate (APS) kinase-catalyzed reaction proceeds through a phosphorylated enzyme intermediate even though both substrates can bind simultaneously to the enzyme. The studies will characterize the functional roles of amino acids located in two sequence motifs: a classic nucleotide binding motif and a motif found in PAPS binding proteins. The serine that is phosphorylated as a reaction intermediate is located between these sequence motifs. Site-directed mutagenesis of the cloned gene for E. coli APS kinase will be followed by in-depth characterization of the mutant enzymes. Novel bi-substrate analogs will be synthesized and tested as inhibitors. These compounds may provide the first potent, selective inhibitors of an enzyme of PAPS biosynthesis, as well as insight into the spatial proximity of the ATP and APS binding sites.
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