ENZYMATIC MECHANISMS OF SULFUR-NUCLEOSIDE METABOLISM

硫-核苷代谢的酶促机制

• 批准号: 3279122
• 负责人: GEORGE Douglas MARKHAM
• 金额: $ 30.08万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279122
• 关键词: Escherichia coli; S adenosylmethionine; bacterial proteins; chemical binding; conformation; divalent cations; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme mechanism; enzyme structure; enzyme substrate; manganese; metalloenzyme; methionine adenosyltransferase; molecular cloning; nuclear magnetic resonance spectroscopy; nucleotide metabolism; phosphotransferases; radiotracer; site directed mutagenesis; vanadium

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.

含硫腺嘌呤核苷衍生物S-腺苷蛋氨酸 和3-磷酸腺苷-5 '-磷酸硫酸盐起着重要作用， 真核和原核细胞的代谢。 这项研究的目的是 的活性中心结构和催化机理 参与这些代谢物生物合成的酶。 这些酶 是S-腺苷甲硫氨酸合成酶（ATP：L-甲硫氨酸S- 腺苷转移酶）和腺苷5 '-磷酸硫酸激酶（ATP： 腺苷5 ′-磷酸硫酸3 ′-磷酸转移酶）。 在这两种情况下的 将研究来自大肠杆菌的酶，因为这些基因是 克隆和分子遗传学的方法可以应用。 对S-腺苷甲硫氨酸合成酶的研究将确定 用稳态动力学方法计算了反应的能量分布。 的 酶结合的S-腺苷甲硫氨酸的构象和动力学将是 通过质子和氘NMR测定。 为了阐明 在两种所需的二价金属离子活化剂中， 活性位点结合的金属离子将通过顺磁共振测定 方法使用Mn（II）和VO 2+作为探针;磁相互作用与 将测量同位素标记的底物和酶。 的 由metX基因产生的单体S-腺苷甲硫氨酸合成酶将被 纯化和表征。 将对克隆的metX基因进行测序， 与已知的metK基因序列进行比较，metK基因编码 四聚体S-腺苷甲硫氨酸合成酶，其先前已被 研究了 特定氨基酸残基在两种化学物质中的作用 将确定在相同活性位点催化的反应 使用克隆的metK基因的随机和定点诱变。 腺苷5 '-磷酸硫酸激酶的研究将决定 使用预稳态动力学的反应中每一步的常数， 同位素捕获实验 磷酸化酶是否在 该反应是一个必不可少的中间体将被确定。 网站 定向诱变将用于去除磷酸化位点，并且 突变酶催化磷酰基转移的能力将 评估。 NMR和EPR研究将用于阐明 磷酸化酶的形成。 光谱研究将揭示 在基材粘合剂之间是否存在实质上的分离 位点，以及E-P的磷酰基基团的迁移率。 活性所需的二价金属离子将由 顺磁共振实验，这将揭示配体的 金属离子和它们之间的距离。