MECHANISM OF ACTIONS OF A MAJOR FOLATE ENZYME

描述：10-甲酰基四氢叶酸脱氢酶(FDH)调节10- 甲酰四氢叶酸(10-甲酰四氢叶酸)和四氢叶酸(THF)池 依赖NADP的脱氢酶将10-甲酰四氢呋喃转化为四氢呋喃和二氧化碳 在NADP非依赖的水解酶反应中反应或生成苏氨酸和甲酸盐。 外佣可能通过控制10-羟色胺的水平来调节嘌呤的生物合成 甲酰四氢呋喃。申请人小组显示，NAP和IMP激活了外籍家庭佣工 这表明存在一个调控的核苷酸结合位点。外佣亦 通过将甲酸盐以二氧化碳的形式去除，在代谢甲酸盐方面起着至关重要的作用， 从而保护细胞免受甲酸盐中毒。这种酶是一种 两种无关蛋白质的自然融合，其中氨基末端 结构域具有底物结合部位和水解酶催化中心 而乙醛脱氢酶同源的催化机理 羧基末端结构域被用来作为催化中心 脱氢酶反应。因此，外佣有两个催化中心，而不是一个 底物结合部位。需要一个100个残基的中间结构域来 将两个功能域结合到中间域是 将两个功能结构域结合在一起以催化 脱氢酶反应。申请人建议中间域 是灵活的，允许构象变化，从而改变 氨基-氨基-末端构象变化的取向 从而改变氨基-氨基-羧基末端结构域的取向 调节酶活性。在体外，外佣建议2-ME直接 参与酶催化机制的具体目的包括：1) 绘制了FDH叶酸结合图，并解析了 FDH氨基末端结构域；2)研究FDH的激活机制 碱基脱氢酶的鉴定和鉴定 3)研究2-ME激活外佣的机制 并寻找可以取代2-ME的天然化合物 在细胞内；以及4)研究中间结构域的作用 FDH在酶功能中的作用。定点突变，截短的表达 FDH的形式、亲和标记、酶活性测定、结合 研究和结晶学方法将用于实现以下目标 这个项目。众所周知，叶酸缺乏和 巨幼细胞性贫血以及最近发现的与 血管疾病的风险以及叶酸在人体内的重要性 神经管缺陷的预防使这些研究变得尤为重要 切合实际。

DESCRIPTION:10-Formyltetrahydrofolate dehydrogenase (FDH) regulates 10- formyltetrahydrofolate (10-formylTHF) and tetrahydrofolate (THF) pools by converting 10-formylTHF to THF and CO2 in an NADP-dependent dehydrogenase reaction or to THR and formate in an NADP-independent hydrolase reaction. FDH may regulate purine biosynthesis by controlling the level of 10- formylTHF. The applicant's group showed that NAP and IMP activate FDH suggesting the existence of a regulatory nucleotide-binding site. FDH also plays an essential role in metabolizing formate by removing it as CO2, thus protecting the cell from formate intoxication. The enzyme is a natural fusion of two unrelated proteins in which the amino-terminal domain bears the substrate-binding site and hydrolase catalytic center while the catalytic machinery of the aldehyde dehydrogenase homologous carboxyl-terminal domain is used as the catalytic center in the dehydrogenase reaction. Thus, FDH has two catalytic centers but one substrate-binding site. A 100-residue intermediate domain is required to bring the two functional domains together to intermediate domain is required to bring the two functional domains together to catalyze the dehydrogenase reaction. The applicant suggest that the intermediate domain of FDH is flexible and allows conformational changes which alter the orientation of the amino- an carboxyl-terminal conformational changes which alter the orientation of the amino-an carboxyl-terminal domains thus regulating enzyme activity. In vitro, FDH proposes that 2-ME be directly involved in the mechanism of enzyme catalysis Specific aims include: 1) to map the FDH folate binding, and to resolve the crystal structure of the amino-terminal domain of FDH; 2) to study the mechanism of activation of FDH by nucleotide including identification and characterization of the regulatory site; 3) to study the mechanism of activation of FDH by 2-ME and to search for naturally occurring compounds which can replace 2-ME inside the cell; and 4) to study the role of the intermediate domain of FDH in enzyme function. Site-directed mutagenesis, expression of truncated forms of FDH, affinity labeling, assay of enzyme activity, binding studies, and crystallographic methods will be used to achieve the goals of the project. The well-known relationship between folate deficiency and megaloblastic anemia a well as the recently discovered association with the risk of vascular disease together with the importance of folate in prevention of neural-tube defects make these studies particularly relevant.