MECHANISM OF ACTION OF A MAJOR FOLATE ENZYME

本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通过依赖nadp的脱氢酶反应或不依赖nadp的水解酶反应将10-甲酰四氢叶酸转化为四氢叶酸，从而调节两个主要的叶酸池。也有人提出，该酶作为细胞内叶酸仓库，保护叶酸辅酶免受氧化降解。这种酶是两种不相关的蛋白质的自然融合。氨基末端结构域包含叶酸结合位点，起水解酶的作用。类乙醛脱氢酶羧基末端结构域与乙醛脱氢酶结合在一个多肽中，作为脱氢酶反应的催化工具。100个残基中间结构域是两个功能结构域之间的连接体，是催化脱氢酶反应所必需的。假设FDH的水解酶反应虽然本身不具有生理意义，但却是FDH脱氢酶机制的重要组成部分。FDH脱氢酶的作用机理是水解酶和醛脱氢酶两个连续反应的结合。在脱氢酶反应过程中，中间产物从FDH的水解酶区域转移到醛脱氢酶区域。中间结构域是使两个功能结构域在正确的方向上进行转移的关键。该项目的另一部分是基于一个假设，即FDH的主要作用之一是通过控制10-甲酰基四氢叶酸水平来调节新的嘌呤生物合成。最近的研究发现，FDH在癌变过程中被高度下调，显然是由于癌细胞对嘌呤的需求增加，这使得该蛋白成为抗癌化疗的重要潜在靶点。提出以下具体目标来检验这些假设。(1)确定中间结构域在酶机制中的作用。(2)表征叶酸结合位点，评价FDH的水解酶作用机制。(3)结晶和解析FDH单个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。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.