ENZYMATIC MECHANISMS OF SULFUR-NUCLEOSIDE METABOLISM

含硫腺嘌呤核苷衍生物S-腺苷蛋氨酸 和腺苷-5'-磷酸硫酸在代谢中起重要作用， 包括真核和原核细胞。 本研究的目的是 表征催化机理和活性中心结构， 催化独特的生物合成反应的酶， 代谢物。 S-腺苷甲硫氨酸合成酶（ATP：L-甲硫氨酸）的研究 S-腺苷转移酶）将决定哪些氨基酸残基是 在底物结合和催化中很重要。 光亲和标记 酶和克隆的结构基因的体外诱变， 将使用酶。 与蛋白质结合的二价金属离子的配体 以及两种金属离子结合的配合物的结构 酶-底物复合物将通过EPR光谱测定。 腺苷甲硫氨酸脱羧酶的研究将确定 酶在具有底物和产物的复合物中的分配 反应的稳定状态。 席夫碱的质子化状态 用15 N NMR表征与底物和产物形成的反应产物。 的 化学治疗剂甲基乙二醛抑制的分子基础 将研究双（鸟苷酰腙）。 是否需要二价 金属离子是变构激活剂，或者如果它在活性位点结合， 被确定。 对ATP硫酸化酶（ATP：sulfate adenylyltransferase）的研究， 确定蛋白质是否结合二价金属离子活化剂， 加入金属-ATP复合物。 平衡常数和 酶结合反应物的相互转化率将按顺序测量 来确定平衡常数是否向1移动 从10到-8的自由反应物。 腺苷-5'-磷酸硫酸激酶的研究 (ATP：腺苷酰硫酸-3'-磷酸转移酶）将研究是否存在一种 二价金属-腺苷-5'-磷酸硫酸盐络合物，以及二价金属-腺苷-5'-磷酸硫酸盐络合物， 金属-ATP复合物为底物。 单价的可能要求 将评估阳离子活化剂。

The sulfur containing adenine nucleoside derivatives S-adenosylmethionine and adenosine-5'-phosphosulfate play essential roles in the metabolsim of both eukaryotic and prokaryotic cells. The objectives of this research are to characterize the catalytic mechanisms and active site structures of enzymes which catalyze unique biosynthetic reactions involving these metabolites. Studies of S-adenosylmethionine (AdoMet) synthetase (ATP:L-methionine S-adenosyltransferase) will determine which amino acid residues are important in substrate binding and catalysis. Photoaffinity labelling of the enzyme and in vitro mutagenesis of the cloned structural gene for the enzyme will be used. The ligands to the protein-bound divalent metal ion and the structures of the complexes with the two metal ions bound in enzyme-substrate complexes will be determined by EPR spectroscopy. The studies of S-adenosylmethionine decarboxylase will determine the partitioning of enzyme among complexes with substrate and product in the steady state of the reaction. The protonation states of the Schiff bases formed with substrate and product will be characterized by 15 N NMR. The molecular basis for inhibition by the chemotherapeutic agent methylglyoxal bis (guanylhydrazone) will be investigated. Whether the required divalent metal ion is an allosteric activator or if it binds at the active site will be determined. The studies of ATP sulfurylase (ATP:sulfate adenylyltransferase) will determine whether the protein binds a divalent metal ion activator, in addition to the metal-ATP complex. The equilibrium constant and interconversion rates for enzyme-bound reactants will be measured in order to determine whether the equilibrium constant is displaced toward unity from the value of 10 to the minus 8 for free reactants. The studies of adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate-3'-phosphotransferase) will investigate whether a divalent metal-adenosine-5'-phosphosulfate complex, as well as a divalent metal-ATP complex, is substrate. The possible requirement for a monovalent cation activator will be assessed.