MECHANISM OF ACTION OF A MAJOR FOLATE ENZYME

该提案的主要目的是了解最丰富的叶酸酶之一10-甲酰四氢叶酸脱氢酶（FDH）的代谢作用和酶促机制。 FDH在NADP依赖性脱氢酶反应或NADP非依赖性水解酶反应中将10-甲酰四氢叶酸转化为四氢叶酸，从而调节两个主要叶酸库。 还提出该酶作为细胞内叶酸贮库保护叶酸辅酶免于氧化降解。 这种酶是两种不相关蛋白质的天然融合体。 氨基末端结构域具有叶酸结合位点并作为水解酶发挥功能。 当醛脱氢酶样羧基末端结构域和羧基末端结构域结合在一个多肽中时，醛脱氢酶样羧基末端结构域作为脱氢酶反应中的催化工具起作用。 一百个残基中间结构域是两个功能结构域之间的接头，需要将它们结合在一起以催化脱氢酶反应。 据推测，FDH的水解酶反应虽然本身没有生理意义，但却是FDH脱氢酶机制的重要和必要部分。 FDH脱氢酶机制是水解酶和醛脱氢酶两个连续反应的组合。 在脱氢酶反应过程中，中间产物从FDH的水解酶结构域转移到醛脱氢酶结构域。 中间结构域对于使两个功能结构域处于正确方向以允许转移至关重要。该项目的另一部分是基于这样的假设，即FDH的主要作用之一是通过控制10-甲酰四氢叶酸水平来调节嘌呤的从头生物合成。最近发现FDH在癌发生中高度下调，显然是由于癌细胞对嘌呤的需求增加，使得该蛋白质成为抗癌化疗中重要的潜在靶点。 为检验这些假设，提出了以下具体目标。 (1)确定中间结构域在酶作用机制中的作用。 (2)研究FDH的叶酸结合位点，探讨FDH的水解机制。 (3)结晶和解析FDH单个结构域和全长蛋白质的晶体结构。 (4)阐明FDH在细胞代谢中的作用。 定点诱变和蛋白质设计方法、酶活性测定、结合研究、晶体学和免疫化学方法、哺乳动物细胞表达、反义寡核苷酸技术、嘌呤和叶酸测定将用于实现该项目的目标。众所周知，叶酸在预防巨幼细胞性贫血、血管疾病、神经管出生缺陷和癌症中的作用使这些研究特别相关。

The broad objectives of this proposal are to understand the metabolic role and enzymatic mechanism of one of the most abundant folate enzymes, 10-formyltetrahydrofolate dehydrogenase (FDH). FDH converts 10-formyltetrahydrofolate to tetrahydrofolate in an NADP-dependent dehydrogenase reaction or in an NADP-independent hydrolase reaction thus regulating two of the major folate pools. It has been also proposed that the enzyme serves as an intracellular folate depot protecting folate coenzymes from oxidative degradation. The enzyme is a natural fusion of two unrelated proteins. The amino-terminal domain bears the folate-binding site and functions as a hydrolase. The aldehyde dehydrogenase like carboxyl-terminal domain works as the catalytic tool in the dehydrogenase reaction when the two domains are combined in one polypeptide. A hundred residue intermediate domain is a linker between the two functional domains required to bring them together to catalyze the dehydrogenase reaction. It is hypothesized that the hydrolase reaction of FDH although by itself is not of physiological significance, is an important and essential part of the FDH dehydrogenase mechanism. The FDH dehydrogenase mechanism is a combination of two sequential reactions, the hydrolase and aldehyde dehydrogenase. During the dehydrogenase reaction transfer of an intermediate product from the hydrolase domain of FDH to the aldehyde dehydrogenase domain takes place. The intermediate domain is crucial to bring two functional domains in correct orientation to allow the transfer. Another part of this project is based on the hypothesis that one of the major roles of FDH is to regulate de novo purine biosynthesis by controlling 10-formyltetrahydrofolate levels. The recent findings that FDH is highly down-regulated in carcinogenesis, apparently due to increased demand of cancer cells for purines, make the protein an important potential target in anticancer chemotherapy. The following specific aims are proposed to test the hypotheses. (1) To determine the role of the intermediate domain in the enzyme mechanism. (2) To characterize the folate binding site and to evaluate the hydrolase mechanism of FDH. (3) To crystallize and to resolve the crystal structure of the FDH individual domains and the full- length protein. (4) To elucidate the role of FDH in cellular metabolism. Site-directed mutagenesis and protein design approaches, enzyme activity assays, binding studies, crystallographic and immunochemical methods, mammalian cell expression, antisense oligonucleotide techniques, purine and folate assays will be used to achieve the goals of the project. The well known role of folate in prevention of megaloblastic anemia, vascular disease, neural tube birth defects and cancer make these studies particularly relevant.