REGULATION OF FOLATE METABOLISM

这项研究的长期目标是描述新陈代谢 叶酸代谢途径在正常和疾病中的调节 并确定依赖叶酸的酶的反应机制。 我们建议继续我们对亚甲基四氢叶酸还原酶的研究， 一种黄素蛋白，调节叶酸代谢中的一个关键分支点。 这种酶催化一个碳单位从 亚甲基四氢叶酸的从头合成途径 甲基，其活性受腺苷蛋氨酸调节。快速 反应和稳态动力学，结合光亲和标记 配体结合位点，将被用来阐明调节的模式 这种酶。我们建议提纯该途径中的下一种酶， 依赖钴胺的甲硫氨酸合成酶的研究 细菌和哺乳动物来源的酶的激活和催化 消息来源。一氧化二氮(N20)对这些酶的抑制作用 常用麻醉剂，也将进行检查。基因编码 对于钴胺依赖和钴胺非依赖性蛋氨酸 来自大肠杆菌的合成酶将被克隆和测序，并为 依赖钴胺的全酶抑制的机制 将寻求合成不依赖钴胺的酶。研究是 还计划研究ZTP的效果，一种细菌警报，它是 信号叶酸剥夺对细胞内两种关键酶的合成 细菌叶酸生物合成、IMP脱氢酶和GTP环水解酶。

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.