FOLINATE UTILIZATION: MECHANISM, REGULATION AND CANCER

依赖于三磷酸腺苷和镁的5-甲酰基四氢叶酸转化为 5，10-亚甲基四氢叶酸催化的5，10-亚甲基衍生物 合成酶(EC 6.3.3.2)是人类生命中仅有的两个反应之一 四氢叶酸介导的一碳代谢利用 以5-甲酰基四氢叶酸为正常底物。这种反应是 在癌症化疗中的重要性，因为它可能为 从大剂量甲氨蝶呤的毒副作用中“拯救”亚叶酸钙 养生法。这一反应代表了一类特殊的 三磷酸腺苷依赖的酶催化酰胺化反应的研究 (在这种情况下，胺和酰胺都位于同一分子上 产生环胺)。对这一事件的详细调查 该酶催化反应的动力学和化学机理 从干酪乳杆菌中提纯到同质将因此提供 对酶催化在这方面的机理的重要洞察 耐人寻味的反应。将特别强调对以下事项的澄清 ATP水解步骤的详细立体化学，因为没有 目前有关于这种类型的ATP依赖反应的信息。这个 三磷酸腺苷水解酶的能量耦合的精确机制 环化反应是不确定的，因为酶催化的环化 异构化的5-甲酰亚胺衍生物不需要三磷酸腺苷或M2。 结合和使用的立体化学要求的表征 M2-ATP络合物的催化作用将用以下方法测定 5‘-硫代核苷及其取代惰性络合物 CR3-ATP和ADP。H4叶酸底物的发色性质 和产品将被用来进行详细的动力学研究 反应机理，并有望获得分光光度证据 形成一种磷酸化中间体。利用所获得的知识 从干酪乳杆菌的酶，将与哺乳动物进行比较 从兔肝中分离出的酶。将进行重点试验，以 确定造成明显差异的结构和机制基础 在动力学机制上，对核苷5‘-三磷酸的亲和力 底物，以及对抑制叶酸衍生物的亲和力。

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.