CHEMISTRY OF FOLATE AND PTERIDINE COENZYMES

这项建议旨在利用在以下方面取得的重大进展 大鼠二氢蝶呤还原酶的结构特征 最后一个支持期。得到了该酶的两种晶型 其分辨率小于2埃，以及apo酶的结构和 表征了一个与NADH形成的二元络合物。此外，DHPR基因 在大肠杆菌中克隆、表达及诱变成功 进行了实验。有了这个基础，它的目的是 产生特定的突变体来定义酶作用的机制和 确认已发现的与黄素酶机制和 短链脱氢酶。所有的突变体都将被定性为 稳定性、比活性和动力学。将尝试创建 通过：(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；以及；。(C)晶体与 最近解析的单斜晶型，包含一个更具溶剂化的 分子结构比早先证明的正交形的分子结构 对这种方法不妥协。人DHPR的cDNA基因序列为 与老鼠密切相关，并将由诱变剂产生 构建，或通过从Lambda噬菌体文库和 通过分子手段快速获得表达的蛋白质晶体结构 更替方法论。最近观察到DHPR的治疗 与cAMP激酶的活性部分一起摄取一种 磷酸/亚基。结构效应将通过以下方式描述 动力学分析的结晶学和力学效应。一名活跃分子 单体将通过突变特定的氨基酸而产生 两亲性疏水四股螺旋束 二聚体形式的单体。这将允许多维核磁共振技术 用于辅助机械分析。它的目的是确定 并将自然发生的遗传错误与患有 具有已知结构的异常PKU，并试图发现 机械性和结构性的颠覆。错误也将被重现 在大肠杆菌表达系统中进行酶的体外检测 行动。它还打算详细说明突变情况，以确定 可持续活动的关键功能区。通过应用图形 对感知到的活跃站点进行分析，意在选择一个 目前尚不存在具有DHPR特异性的抑制剂。它的目的还在于 进一步探索我们最初有趣的实验，将DHPR提供给 胞浆通过蛋白结合的叶酸和叶酸结合蛋白 细胞膜。 表面相似的二氢叶酸还原酶的两个方面 (Dhfr)也有待探索。在一种情况下，“适配子”技术 将被用于检测存在于 原核和真核来源的抗叶酸抑制物结合部位 酶作为证明适体技术可行性的模型，以及 其次，分枝杆菌DHFRs被忽视的领域将通过 该酶的分离及其结构和机理研究 来自结核分枝杆菌和禽类分枝杆菌。

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.