FOLINATE UTILIZATION: MECHANISM, REGULATION AND CANCER

叶酸的利用：机制、调节和癌症

• 批准号: 3183861
• 负责人: CHARLES EDWARD GRIMSHAW
• 金额: $ 10.73万
• 依托单位: SCRIPPS CLINIC AND RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 1990-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3183861
• 关键词: Lactobacillus casei; adenosine triphosphate; chemical binding; enzyme structure; enzyme substrate; fluorescence spectrometry; leucovorin; ligase; liver; magnesium; neoplasm /cancer chemotherapy; phosphodiesterases; tetrahydrofolates

The ATP- and Mg2+-dependent conversion of 5-formyltetrahydrofolate to the 5,10-methenyl derivative, catalyzed by 5,10-methenyltetrahydrofolate synthetase (EC 6.3.3.2) is one of only two reactions in tetrahydrofolate-mediated one-carbon metabolism which utilize 5-formyltetrahydrofolate as the normal substrate. This reaction is of importance in cancer chemotherapy, since it may provide the basis for "leucovorin-rescue" from the deleterious effects of high-dose Methotrexate regimens. The reaction is representative of a special class of ATP-dependent enzymes catalyzing the formation of an amidine from an amide (in this case both the amine and amide reside on the same molecule resulting in a cyclic amidinium). A detailed investigation into the kinetic and chemical mechanism of the reaction catalyzed by the enzyme purified to homogeneity from Lactobacillus casei will thus provide significant insight into the mechanism of enzymic catalysis in this intriguing reaction. Particular emphasis will be applied to elucidation of the detailed stereochemistry of the ATP hydrolysis step, since no information currently exists for this type of ATP-dependent reaction. The precise mechanism for coupling of the energy of ATP hydrolysis to the cyclization reaction is uncertain, since enzyme-catalyzed cyclization of the isosteric 5-formimino derivative shows no requirement for ATP or M2+. Characterization of the stereochemical requirements for binding and catalysis of the M2+-ATP chelate complex will be determined using nucleoside 5'-phosphorothioates and substitution-inert chelate complexes of Cr3+-ATP and ADP. The chromophoric properties of the H4folate substrate and product will be utilized to conduct detailed kinetic studies of the reaction mechanism, and hopefully obtain spectrophotometric evidence for formation of a phosphorylated intermediate. Using the knowledge gained from the L. casei enzyme, a comparison will be made with the mammalian enzyme isolated from rabbit liver. Key experiments will be conducted to determine the structural and mechanistic basis for the apparent differences in kinetic mechanism, affinity for the nucleoside 5'-triphosphate substrate, and affinity for inhibitory folate derivatives.

5-甲酰四氢叶酸依赖于ATP和Mg 2+转化为 5，10-亚甲基衍生物，由5，10-亚甲基四氢叶酸催化 合成酶（EC 6.3.3.2）是在生物合成中仅有的两种反应之一。 四氢叶酸介导一碳代谢， 5-甲酰四氢叶酸作为正常底物。 这种反应是 在癌症化疗中的重要性，因为它可以提供基础， 从大剂量甲氨蝶呤的有害作用中“甲酰四氢叶酸-拯救” 养生法 该反应代表了一类特殊的 催化酰胺形成脒的ATP依赖性酶 (in在这种情况下，胺和酰胺都位于同一分子上 产生环状脒鎓）。 详细调查了 酶催化反应的动力学和化学机理 因此，从干酪乳杆菌中纯化至均质， 对酶催化机制的重要见解， 有趣的反应 特别强调的是， ATP水解步骤的详细立体化学，因为没有 目前存在这种类型的ATP依赖性反应的信息。 的 ATP水解的能量耦合到 环化反应是不确定的，因为酶催化的环化 电子等排的5-甲亚氨基衍生物显示不需要ATP或M2+。 表征结合的立体化学要求， M2+-ATP螯合复合物的催化作用将使用 核苷5 ′-硫代磷酸酯和取代惰性螯合物 Cr 3 +-ATP和ADP。 H4叶酸底物的发色性质 和产品将用于进行详细的动力学研究， 反应机理，并希望获得分光光度证据 磷酸化中间体的形成。 利用获得的知识 从L.酪蛋白酶，将与哺乳动物的 从兔子肝脏中分离的酶。 将进行关键实验， 确定明显差异的结构和机制基础 在动力学机制中，对核苷5 '-三磷酸的亲和力 底物和对抑制性叶酸衍生物的亲和力。