ENZYMATIC MECHANISMS OF SULFUR NUCLEOSIDE METABOLISM

性状（改编自申请人摘要）S-腺苷甲硫氨酸（蛋氨酸） 是主要的生物烷基化剂， 所有细胞的新陈代谢。因此，参与依赖于HDMet的酶 过程是开发化疗剂的目标。的 本研究的目的是阐明活性位点结构， 这两种酶的催化机制。 对ATP：L-甲硫氨酸S-腺苷转移酶的研究将 阐明了新发现的高亲和力抑制的结构基础， 慢结合抑制剂，中间体类似物二亚氨基三磷酸 （O3 P-NH-PO2-NH-PO3）。动力学和光谱学研究将揭示 酶-抑制剂复合物的形成以及结合的结构 抑制剂.蛋氨酸合成酶的晶体结构显示了一个灵活的环 这是一个重要的催化过程。的 环序列的长度和组成对催化活性的影响 功能将被公开。循环的动力学特征如下： 自旋标记环残基的EPR光谱;无论是底物还是产物 将阐明改变环路动力学。产自拟南芥的蛋氨酸合成酶 与真核生物和原核生物有着非常不同的序列， 这表明了一种改变的催化策略;詹氏甲烷球菌 合成酶将被表征。预测的催化性能差异 将使用野生型的结构和功能研究进行评估 酶和选择的突变体。 蛋氨酸脱羧酶催化将产物引导至 多胺生物合成这种酶含有一种不寻常的共价键， 丙酮酸基团，与底物反应形成席夫碱 中间体对E.大肠杆菌，这需要一个 二价金属离子的活性和来自M. 将阐明Jannaschii。本文研究了反应器中各步反应的速率和平衡， 机理将由稳态动力学方法确定。活性 游离酶和复合物中的位点结构将通过NMR表征， 富含13 C-丙酮酸的酶和富含13 C和15 N的底物。磁 Mn 2+络合物的共振研究将揭示二价金属离子是否 激活剂在活性位点结合，可能与丙酮酸配位， 促进Schiff碱的形成。本文还探讨了抑制作用的机理 化学治疗剂甲基乙二醛双（鸟苷酰腙）将通过 动力学和NMR测量，揭示抑制是否来自 与乙酰基部分形成共价加合物。

DESCRIPTION (adapted from applicant's abstract) S-adenosylmethionine (AdoMet) is the primary biological alkylating agent and occupies an essential role in the metabolism of all cells. Thus, enzymes involved in AdoMet dependent processes are targets for development of chemotherapeutic agents. The objectives of this research are to elucidate the active site structures and catalytic mechanisms of two of these enzymes. Studies of AdoMet synthetase (ATP:L-methionine S-adenosyltransferase) will elucidate the structural basis for inhibition by a newly found high affinity, slow binding inhibitor, the intermecliate analog diimidotriphosphate (O3P-NH-PO2-NH-PO3). Kinetic and spectroscopic studies will reveal the steps in formation of the enzyme-inhibitor complex, and the structure of the bound inhibitor. The crystal structure of AdoMet synthetase shows a flexible loop which gates access to the active site, an important catalytic event. The influence of the length and composition of the loop sequence on catalytic function will be unveiled. The dynamics of the loop will be characterized by EPR spectroscopy of a spin-labeled loop residue; whether substrates or products alter the loop dynamics will be elucidated. AdoMet synthetases from Archaea have very different sequences from those of the eukarya and prokarya, suggesting an altered catalytic strategy; the Methanococcus jannaschii synthetase will be characterized. Predicted differences in catalytic properties will be evaluated using structural and functional studies of the wild type enzyme and selected mutants. AdoMet decarboxylase catalyzes the reaction that directs the product to polyamine biosynthesis. The enzyme contains an unusual covalently attached pyruvate group that forms a Schiff base with the substrate as a reaction intermediate. The mechanisms of the enzyme from E. coli, which requires a divalent metal ion for activity and the metal independent enzyme from M. jannaschii will be elucidated. The rates and equilibria of the steps in the mechanism will be determined by presteady state kinetic methods. The active site structure in the free enzyme and complexes will be characterized by NMR of l3C-pyruvate enriched enzyme, and 13C and l5N enriched substrate. Magnetic resonance studies of Mn2+ complexes will reveal whether the divalent metal ion activator binds at the active site, perhaps coordinating the pyruvate to facilitate the Schiff base formation. The mechanism of inhibition by the chemotherapeutic agent methylglyoxal bis(guanylhydrazone) will be determined by kinetic and NMR measurements, revealing whether inhibition results from formation of a covalent adduct with the pyruvyl moiety.