REGULATION OF FOLATE METABOLISM

这项研究的长期目标是表征代谢 调节正常和患病患者的叶酸代谢途径 状态，并确定叶酸依赖酶的反应机制。 我们建议继续我们对亚甲基四氢叶酸还原酶的研究， 一种调节叶酸代谢关键分支点的黄素蛋白。 这种酶催化一个碳单元从一个碳单元到另一个碳单元的流动的第一步。 亚甲基四氢叶酸进入从头生物合成途径 甲基，其活性受腺苷甲硫氨酸调节。 快速 反应和稳态动力学，再加上光亲和标记的 配体结合位点，将被用来阐明调控模式， 这种酶。 我们建议纯化途径中的下一个酶， 钴胺素依赖的蛋氨酸合酶，并研究 来自细菌和哺乳动物的酶的活化和催化 源 一氧化二氮（N20）对这些酶的抑制作用， 常用的麻醉剂，也将被检查。 基因编码 对于钴胺素依赖性和钴胺素非依赖性蛋氨酸 合成酶;大肠杆菌将被克隆和测序， 钴胺素依赖性全酶抑制 将寻求钴胺素非依赖性酶的合成。 研究是 还计划研究ZTP的作用，ZTP是一种细菌报警素， 信号叶酸剥夺，对合成的两个关键酶， 细菌叶酸生物合成、IMP脱氢酶和GTP环化水解酶。

The long term goal of this research is to characterize the metabolic regulation of pathways of folate metabolism in both normal and diseased states, and to determine reaction mechanisms for folate dependent-enzymes. We propose to continue our studies on methylenetetrahydrofolate reductase, a flavoprotein which regulates a key branch point in folate metabolism. This enzyme catalyzes the first step in the flow of one carbon units from methylenetetrahydrofolate into the pathway for de novo biosynthesis of methyl groups, and its activity is regulated by adenosylmethionine. Rapid reaction and steady-state kinetics, coupled with photoaffinity labelling of ligand binding sites, will be used to elucidate the mode of regulation of this enzyme. We propose to purify the next enzyme in the pathway, cobalamin-dependent methionine synthase, from pig liver and to study the activation of and catalysis by enzymes from both bacterial and mammalian sources. The inhibition of these enzymes by nitrous oxide (N20), a commonly used anaesthetic agent, will also be examined. The genes coding for both the cobalamin-dependent and cobalamin-independent methionine synthase enzymes from E. coli will be cloned and sequenced and evidence for the mechanism by which the cobalamin-dependent holoenzyme represses synthesis of the cobalamin-independent enzyme will be sought. Studies are also planned to investigate the effect of ZTP, a bacterial alarmone which signals folate deprivation, on the synthesis of two key enzymes in bacterial folate biosynthesis, IMP dehydrogenase and GTP cyclohydrolase.