ENZYMATIC MECHANISMS OF SULFUR NUCLEOSIDE METABOLISM

含硫腺嘌呤核苷衍生物S-腺苷蛋氨酸 和3-磷酸腺苷-5‘-磷酸硫酸盐在 真核和原核细胞的代谢。这项研究的目标是 都是为了阐明两个化合物的活性中心结构和催化机理 参与这些代谢物的生物合成的酶。这些酶 是S-腺苷蛋氨酸合成酶(三磷酸腺苷：L-蛋氨酸S- 腺苷转移酶)和腺苷5‘-磷酸硫酸盐激酶(ATP： 腺苷5‘-磷酸-硫酸盐3’-磷酸转移酶)。在这两种情况下， 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都可以应用。 S-腺苷甲硫氨酸合成酶的研究将确定完全游离 用稳态前动力学方法计算反应的能量分布。这个 酶结合的S-腺苷蛋氨酸的构象和动力学 由质子和重氢核磁共振确定。为了阐明机械论的作用 在两种必需的二价金属离子活化剂中， 活性位结合金属离子将通过顺磁共振确定 方法使用Mn(II)和VO2+作为探针； 将测量同位素标记的底物和酶。这个 甲基X基因产生的单体S-腺苷甲硫氨酸合成酶 提纯和鉴定。将对克隆的metX基因进行测序 与已知的编码该基因的metK基因序列的比较 四聚体S-腺苷蛋氨酸合成酶 学习。特定氨基酸残基在这两种化学物质中的作用 在同一活性中心催化的反应将被确定 对克隆的metK基因进行随机和定点诱变。 对腺苷5‘-磷酸硫酸盐激酶的研究将决定这一比率 反应中每一步的常数使用稳态态前动力学和 同位素捕获实验。磷酸化酶是否形成于 反应是一种必须的中间体，将予以确定。立地 定向突变将被用来去除磷酸化位点，以及 突变酶催化磷酸转移的能力将是 已评估。核磁共振和EPR研究将被用来阐明 形成一种磷酸化的酶。光谱研究将揭示 衬底结合之间是否有实质性的分离 和E-P的磷酸基的迁移率。政府官员的角色 活性所需的二价金属离子将从 顺磁共振实验将揭示配体到 金属离子和它们之间的距离。

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.