MECHANISM OF ACTIONS OF A MAJOR FOLATE ENZYME

主要叶酸酶的作用机制

• 批准号: 2906276
• 负责人: SERGEY A KRUPENKO
• 金额: $ 1.72万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2906276
• 关键词: NAD(H) phosphate; active sites; affinity labeling; crystallization; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; epitope mapping; folate; high performance liquid chromatography; nicotinamide adenine dinucleotide; nucleotides; oxidoreductase; protein binding; reducing agents; site directed mutagenesis; thiols; transfection /expression vector; vitamin metabolism

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.

描述：10种甲基四氢叶酸脱氢酶（FDH）调节10- 原肌四氢叶酸（10型基团）和四氢叶酸盐（THF）池通过 将10型甲基酰基转换为NADP依赖性脱氢酶中的THF和CO2 反应或在非依赖NADP的水解酶反应中的THR和甲酸盐。 FDH可以通过控制10-的水平来调节嘌呤的生物合成 emylthf。申请人的小组表明午睡和激活FDH 表明存在调节性核苷酸结合位点。也是FDH 通过将其删除为CO2，在代谢甲酸甲酸甲酸甲酸甲酸甲酸甲酸甲酸盐中起着至关重要的作用 从而保护细胞免受甲酸中毒的侵害。酶是 两种无关蛋白质的自然融合，其中氨基末端 域带有底物结合位点和水解酶催化中心 而醛脱氢酶同源的催化机械 羧基末端结构域用作催化中心 脱氢酶反应。因此，FDH有两个催化中心，但一个 底物结合位点。需要一个100个残留的中间域才能 将两个功能域聚集到中间域IS 需要将两个功能域聚集在一起以催化 脱氢酶反应。申请人建议中间域 FDH的灵活性，允许构象变化改变 氨基的方向 - 羧基末​​端构象变化 这改变了氨基羧基末端结构域的方向 调节酶活性。在体外，FDH提出2-Me直接 参与酶催化特定目的的机理包括：1） 绘制FDH叶酸结合，并解析 FDH的氨基末端域； 2）研究激活机制 FDH通过核苷酸，包括鉴定和表征 监管地点； 3）研究2-Me激活FDH的机制 并搜索可以替代2-Me的自然发生的化合物 在牢房内； 4）研究中间领域的作用 酶功能中的FDH。位置定向诱变，截短的表达 FDH的形式，亲和力标记，酶活性测定，结合 研究和晶体学方法将用于实现目标 项目。叶酸不足与 巨大细胞贫血是最近发现的与 血管疾病的风险以及叶酸在 预防神经管缺陷使这些研究特别 相关的。