ENZYMATIC MECHANISMS OF SULFUR NUCLEOSIDE METABOLISM

说明(改编自申请者摘要)S-腺苷蛋氨酸(ADOMet) 是主要的生物烷基化试剂，在 所有细胞的新陈代谢。因此，参与ADOMet的酶依赖于 工艺是化疗药物开发的目标。这个 这项研究的目的是阐明活性中心的结构和 其中两种酶的催化机制。 ADOMet合成酶的研究(三磷酸腺苷：L-蛋氨酸S-腺苷转移酶) 阐明新发现的高亲和力抑制的结构基础， 慢结合缓蚀剂--中间体类似物二亚胺三磷酸 (O3P-NH-PO2-NH-PO3)。动力学和光谱研究将揭示 酶-抑制物复合体的形成和结合的结构 抑制剂。ADOMet合成酶的晶体结构呈柔性环 它开启了通往活性部位的通道，这是一个重要的催化事件。这个 环序列的长度和组成对催化性能的影响 功能将揭开面纱。循环的动态将由以下几个方面来描述 自旋标记的环残基的EPR波谱；无论是底物还是产物 改变环路动力学将被阐明。古生代的ADOMet合成酶 与真卡雅人和普罗卡亚人的序列非常不同， 暗示了一种改变的催化策略；詹纳斯基甲烷球菌 将对合成酶进行表征。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 酶和选定的突变体。 ADOMet脱羧酶催化反应，将产物导向 多胺生物合成。这种酶含有一种不同寻常的共价连接 与底物反应形成席夫碱的丙酮酸基团 中级的。来自大肠杆菌的酶的机制，这需要一个 二价金属离子的活性和M。 Jannaschii将被澄清。模型中步骤的速度和平衡点 机理将由稳态前动力学方法确定。主动者 游离酶和络合物中的位置结构将通过核磁共振进行表征 L3C-丙酮酸富集酶，13C和15N富集酶。磁性 对Mn2+络合物的共振研究将揭示二价金属离子 激活剂结合在活性部位，可能协调丙酮酸 促进席夫碱基的形成。的抑制机制。 化疗剂甲基乙醛双(鸟酰肼)的测定 动力学和核磁共振测量揭示了抑制是否会导致 与丙酮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.