REGULATION OF FOLATE METABOLISM

这项研究的长期目标是阐明催化作用 利用四氢叶酸衍生物作为辅因子的酶的机制， 并研究一种碳代谢的调节。这项研究将 重视钴胺依赖两种酶的作用机制研究 蛋氨酸合成酶和丝氨酸羟甲基转移酶，这是潜在的 癌症化疗的靶点，但没有特定的抑制物 目前可用。提出了具体的设计策略 这些酶中每一种的活性位点定向抑制物。钴胺- 依赖蛋氨酸合成酶催化甲基从 甲基四氢叶酸(CH3-H4叶酸)转化为同型半胱氨酸，与酶- 结合钴胺假体基团在甲基中的中介作用 调职。我们计划确定底物CH3- 利用紫外可见光谱和核磁共振技术对H4叶酸进行甲基转移活化 确定底物是质子化还是氧化的光谱分析 在死胡同的三元络合物中。我们将调查其中的机制 这种酶被常用的麻醉剂一氧化二氮灭活， 并将尝试设计能够使 酶的作用机制与此类似。关于激活剂的相关研究 甲硫氨酸合成酶的还原甲基化及其催化作用 一种钴胺非依赖性蛋氨酸合成酶的作用机制 还提出了非常相似的反应。我们将继续我们的学习 吡哆醛丝氨酸羟甲基转移酶催化机理的研究 催化β-碳转移的依赖于磷酸的酶 将丝氨酸转化为H4叶酸，生成甘氨酸和CH2-H4叶酸。这些研究 将试图阐明H4叶酸在反应机理中的作用 并将探索使用卤化氨基酸作为潜在的抑制剂 催化作用的作用。对两者都有高度特异性的抑制剂的可用性 蛋氨酸合成酶和丝氨酸羟甲基转移酶不仅会 建议针对这些酶的化疗靶向的方法，但 也将允许评估这些酶在细胞中的作用 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.