REGULATION OF FOLATE METABOLISM

这项研究的长期目标是阐明催化 使用四氢叶酸衍生物作为辅因子的酶的机制， 研究一碳代谢的调控。 这项研究将 强调两种酶的机制研究，钴胺素依赖性 蛋氨酸合酶和丝氨酸羟甲基转移酶，这是潜在的 癌症化疗的靶点，但没有特异性抑制剂， 目前可用。 提出了具体的设计策略 这些酶的活性位点定向抑制剂。 钴胺素- 依赖性甲硫氨酸合酶催化甲基从 甲基四氢叶酸（CH 3-H4叶酸）与同型半胱氨酸，与酶- 作为甲基化中间体的结合钴胺素辅基 转移 我们计划确定底物CH 3- 通过使用UV-可见光和NMR活化H4叶酸进行甲基转移 光谱学以确定底物是质子化的还是氧化的 在死端三元复合物中。 我们将研究 酶被常用的麻醉剂，一氧化二氮， 并将尝试设计能够灭活 酶通过类似的机制。 激活的相关研究 甲硫氨酸合酶的还原甲基化，并对催化 钴胺素非依赖性甲硫氨酸合酶催化 还提出了非常相似的反应。 我们将继续我们的研究 丝氨酸羟甲基转移酶的催化机制， 催化β-碳转移的磷酸盐依赖性酶 丝氨酸转化为H4叶酸，得到甘氨酸和CH 2-H4叶酸。 这些研究 将试图阐明H4叶酸在反应机制中的作用 并将探索使用卤代氨基酸作为潜在的抑制剂 催化作用。 高度特异性抑制剂的可用性， 蛋氨酸合酶和丝氨酸羟甲基转移酶不仅 提出了这些酶的化疗靶向方法，但是 还将允许评估这些酶在细胞内的作用， 新陈代谢.

The long term goals of this research are to elucidate the catalytic mechanisms of enzymes that use tetrahydrofolate derivatives as cofactors, and to study the regulation of one carbon metabolism. This research will emphasize mechanistic studies of two enzymes, cobalamin-dependent methionine synthase and serine hydroxymethyltransferase, that are potential targets for cancer chemotherapy, but for which no specific inhibitors are currently available. Strategies are proposed for the design of specific active site-directed inhibitors of each of these enzymes. Cobalamin- dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate (CH3-H4folate) to homocysteine, with the enzyme- bound cobalamin prosthetic group serving as an intermediary in the methyl transfer. We plan to determine the mode by which the substrate, CH3- H4folate, is activated for methyl transfer by using UV-visible and NMR spectroscopy to determine whether the substrate is protonated or oxidized in dead-end ternary complexes. We will investigate the mechanism by which the enzyme is inactivated by the commonly used anesthetic, nitrous oxide, and will attempt to design agents that are capable of inactivation of the enzyme by a similar mechanism. Related studies on the activation of methionine synthase by reductive methylation, and on the catalytic mechanism of a cobalamin-independent methionine synthase that catalyzes a closely similar reaction are also proposed. We will continue our studies on the catalytic mechanism of serine hydroxymethyltransferase, a pyridoxal phosphate-dependent enzyme that catalyzes the transfer of the beta-carbon of serine to H4folate to yield glycine and CH2-H4folate. These studies will attempt to elucidate the role of H4folate in the reaction mechanism and will explore the use of halogenated amino acids as potential inhibitors of catalysis. The availability of highly specific inhibitors for both methionine synthase and serine hydroxymethyltransferase will not only suggest approaches for chemotherapeutic targeting of these enzymes, but will also permit an assessment of the role of these enzymes in cellular metabolism.