MECHANISM OF ACTION OF A MAJOR FOLATE ENZYME

这项建议的广泛目标是了解最丰富的叶酸酶之一，10-甲酰基四氢叶酸脱氢酶(FDH)的代谢作用和酶机制。FDH在NADP依赖的脱氢酶反应或NADP非依赖的水解酶反应中将10-甲酰基四氢叶酸转化为四氢叶酸，从而调节两个主要的叶酸库。也有人提出，该酶作为细胞内的叶酸库，保护叶酸辅酶免受氧化降解。这种酶是两种无关蛋白质的自然融合。氨基末端含有叶酸结合部位，具有水解酶的功能。当两个结构域结合成一个多肽时，醛脱氢酶类似于羧基末端结构域在脱氢酶反应中起催化作用。一百个残基中间结构域是两个功能结构域之间的连接物，这两个功能结构域需要将它们结合在一起来催化脱氢酶反应。推测FDH的水解酶反应虽然本身并不具有生理意义，但却是FDH脱氢酶机制中一个重要而必要的部分。FDH脱氢酶的作用机制是水解酶和乙醛脱氢酶两个连续反应的组合。在脱氢酶反应中，中间产物从FDH的水解酶结构域转移到乙醛脱氢酶结构域。中间结构域对于使两个功能结构域处于正确的方向以允许转移至关重要。这个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-甲酰四氢叶酸的水平来调节从头合成的嘌呤。最近的研究发现，FDH在致癌过程中被高度下调，这显然是由于癌细胞对嘌呤的需求增加，使该蛋白成为抗癌化疗的重要潜在靶点。为了检验这些假设，本文提出了以下具体目标。(1)确定中间结构域在酶机制中的作用。(2)研究FDH的叶酸结合部位，探讨FDH的水解酶机制。(3)结晶并解析FDH各结构域和全长蛋白的晶体结构。(4)阐明FDH在细胞代谢中的作用。将使用定点突变和蛋白质设计方法、酶活性分析、结合研究、结晶学和免疫化学方法、哺乳动物细胞表达、反义寡核苷酸技术、嘌呤和叶酸分析来实现该项目的目标。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方面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.