CHEMISTRY OF FOLATE AND PTERIDINE COENZYMES

叶酸和蝶啶辅酶的化学性质

• 批准号: 2085985
• 负责人: JOHN M WHITELEY
• 金额: $ 23.83万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-05-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2085985
• 关键词: Escherichia coli; NAD(H) analog; X ray crystallography; affinity chromatography; chemical binding; chemical substitution; chemical synthesis; cofactor; dihydrofolate reductase; enzyme inhibitors; enzyme mechanism; enzyme structure; fluorescent dye /probe; folate; folate antagonist; gene expression; histochemistry /cytochemistry; human tissue; laboratory rat; nuclear magnetic resonance spectroscopy; nucleic acid sequence; oxidoreductase; polymerase chain reaction; protein engineering; pteridine analog; pteridines; site directed mutagenesis; thymidylate synthase; vitamin analog

This proposal seeks to exploit the significant advances made in structurally characterizing rat dihydropteridine reductase (DHPR) during the last support period. Two crystal forms of the enzyme were obtained with resolution less than 2 Angstroms and structures of the apo enzyme and a binary complex with NADH were characterized. In addition, the DHPR gene was cloned and expressed in Escherichia coli and successful mutagenesis experiments were performed. With this foundation it is intended to generate specific mutants to define the mechanism of enzymatic action and confirm discovered similarities to flavin enzyme mechanisms and to those of the short-chain dehydrogenases. All mutants will be characterized for stability, specific activity and kinetics. Attempts will be made to create a ternary complex by: (a) using molecular graphics; (b) specific mutants having substrate affinity but no activity, and; (c) crystal soaks with the recently resolved monoclinic crystal form that contains a more solvated molecular structure than the orthorhombic form that has earlier proven intransigent to this approach. The human DHPR cDNA gene sequence is closely related to the rat and will be generated either by mutagenic construction, or by PCR techniques from a lambda phage library and the expressed protein crystal structure will be rapidly obtained by molecular replacement methodology. It was recently observed that treatment of DHPR with the active fraction from cAMP kinase gives uptake of one phosphate/subunit. The structural effects will be delineated by crystallography and mechanistic effects by kinetic analyses. An active monomer will be created by mutating specific amino acids that contribute to the amphipathic hydrophobic four helix bundle that holds the two monomers in dimeric form. This will allow multidimensional NMR techniques to be used to assist in mechanistic analysis. It is intended to determine and correlate naturally occurring genetic errors from patients with aberrant PKU with the known structure and attempt to discover patterns of mechanistic and structural disruption. The errors will also be reproduced in the E. coli expression system for in vitro examination of enzyme action. It is also intended to elaborate the mutant picture to determine the key functional regions for viable activity. By applying graphics analysis to the perceived active site, it is intended to select an inhibitor with DHPR specificity as none yet exist. It is also intended to explore further our initial intriguing experiments to deliver DHPR to the cytosol via protein-bound folate and the folate binding protein of the cellular membrane. Two aspects of the superficially similar enzyme dihydrofolate reductase (DHFR) are also to be explored. In one instance the 'aptamer' technique will be employed to detect the known distinction that exists between the antifolate inhibitor sites of prokaryotic and eukaryotic sources of this enzyme as a model for proving the feasibility of aptamer technology, and secondly the neglected field of mycobacterial DHFRs will be probed by the isolation and structural and mechanistic characterization of this enzyme from Mycobacterium tuberculosis and Mycobacterium avium.

该提案旨在利用在以下方面取得的重大进展： 大鼠二氢蝶啶还原酶（DHPR）的结构表征 最后一次支持。得到了两种晶型的酶 分辨率小于2埃，载脂蛋白酶的结构， 一个二元复合物与NADH的特点。此外，DHPR基因 在大肠杆菌中克隆表达并成功诱变 进行了实验。有了这个基础， 产生特定的突变体以确定酶促作用的机制， 证实发现的相似性黄素酶机制和那些 短链淀粉酶的基因所有突变体将被表征为 稳定性、比活性和动力学。将尝试创建 三元复合物：（a）使用分子图形;（B）特定突变体 具有底物亲和力但没有活性，并且;（c）晶体被底物浸泡 最近解析的单斜晶型，含有更溶剂化的 分子结构的正交形式，已较早证明 对这种做法不妥协。人DHPR cDNA基因序列是 与大鼠密切相关，将由诱变剂 构建，或通过来自λ噬菌体文库的PCR技术， 表达的蛋白质晶体结构将通过分子生物学方法快速获得， 替换方法。最近观察到DHPR的治疗 与来自cAMP激酶的活性级分结合， 磷酸盐/亚基。结构性影响将由 晶体学和动力学分析的机械效应。积极 单体将通过突变特定的氨基酸产生， 两亲疏水的四螺旋束， 二聚体形式的单体。这将使多维核磁共振技术 用于辅助机械分析。它旨在确定 并将患者自然发生的遗传错误与 已知结构的异常PKU，并试图发现 机械性和结构性的破坏。错误也将被复制 在急诊一种用于酶体外检测的大肠杆菌表达系统 行动上它还打算详细说明突变体的情况，以确定 关键的功能区域。通过应用图形 通过对感知到的活性位点的分析， 具有DHPR特异性抑制剂，因为还不存在。它还旨在 进一步探索我们最初有趣的实验，将DHPR传递到 细胞溶质通过蛋白质结合的叶酸和叶酸结合蛋白的 细胞膜 二氢叶酸还原酶的两个方面 （DHFR）也将被探索。在一个例子中，“适体”技术 将被用来检测存在于 抗叶酸抑制剂位点的原核和真核来源， 酶作为证明适体技术可行性的模型，以及 其次，被忽视的分枝杆菌DHFR领域将由 该酶分离和结构及机理表征 来自结核分枝杆菌和鸟分枝杆菌。