The sulfur containing adenine nucleoside derivatives S-adenosylmethionine and 3-phosphoadenosine-5'-phosphosulfate play essential roles in the metabolism of eukaryotic and prokaryotic cells. The goals of this research are to elucidate the active site structures and catalytic mechanisms of two enzymes involved in the biosynthesis of these metabolites. These enzymes are S-adenosylmethionine synthetase (ATP:L-methionine S- adenosyltransferase) and adenosine 5'-phosphosulfate kinase (ATP: adenosine 5'-phosphosulfate 3'-phosphotransferase). In both cases the enzymes from Escherichia coli will be investigated since the genes are cloned and the methods of molecular genetics can be applied. Studies of S-adenosylmethionine synthetase will determine the complete free energy profile for the reaction by presteady state kinetic methods. The conformation and dynamics of enzyme-bound S-adenosylmethionine will be determined by proton and deuterium NMR. To elucidate the mechanistic roles of the two required divalent metal ion activators, the ligands to the active site bound metal ions will be determined by paramagnetic resonance methods using Mn(II) and VO2+ as probes; magnetic interactions with isotopically labelled substrates and enzyme will be measured. The monomeric S-adenosylmethionine synthetase produced by the metX gene will be purified and characterized. The cloned metX gene will be sequenced for comparison with the known sequence of the metK gene which codes for the tetrameric S-adenosylmethionine synthetase that has previously been studied. The roles for particular amino acid residues in the two chemical reactions which are catalyzed at the same active site will be determined using random and site directed mutagenesis of the cloned metK gene. Studies of adenosine 5'-phosphosulfate kinase will determine the rate constants for each step in the reaction using presteady state kinetics and isotope trapping experiments. Whether the phosphorylated enzyme formed in the reaction is an obligatory intermediate will be determined. Site directed mutagenesis will be used to remove the phosphorylation site, and the ability of the mutant enzyme to catalyze phosphoryl transfer will be evaluated. NMR and EPR studies will be used to elucidate the rationale for formation of a phosphorylated enzyme. Spectroscopic studies will reveal whether there is substantial separation between the substrate binding sites, and the mobility of the phosphoryl group of E-P. The roles of the divalent metal ions which are required for activity will be determined from paramagnetic resonance experiments which will reveal the ligands to the metal ions and the distance between them.
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