CHEMISTRY OF FOLATE AND PTERIDINE COENZYMES

作为蝶呤/叶酸化学的既定计划的一部分 在酶学方面，这项建议主要集中在两个方面，即比较 大鼠肝脏二氢蝶呤及其作用机制的研究 二氢叶酸还原酶(DHPR和DHFR)及其化学 叶酸/蝶啶类似物可作为机械探针或 叶酸/蝶呤代谢中的酶抑制剂，并可能 也具有化疗潜力。这两种还原酶是 重要的是因为它们参与代谢周期(嘌呤， 嘧啶和儿茶酚胺的生物合成)。DHPR 已经得到结晶，重原子衍生品已经 进行了初步的X射线结晶学测量 作为获得完整的三维图像的前奏 结构。为了帮助实现这一目标，已经克隆了这种酶 并进行了测序。在下一个项目期间，它的目的是 完成X光工作，并与 已知的哺乳动物DHFR结构与结合位点，同源性， 可能的进化分歧和比较反应 机械装置。为协助实现这些目标，卫生与公众服务部 基因将在大肠杆菌中表达，并具有位点特异性 将引入突变来确定配体-宿主 关键氨基酸在酶结合部位的相互作用。这个 信息将被用来预测蝶呤结构类似物 可能会强烈抑制DHPR，因为到目前为止还没有紧密的结合 这种酶的抑制剂是已知的。这样的分子将是 对于检查DHPR的新陈代谢非常宝贵，并可能有助于发现 其无处不在的组织分布的原因 与芳香氨基酸羟基酶的协同作用是 仅见于肝脏、肾脏和神经组织。来补充 DHPR核苷酸结合位点的整体结构分析 将使用放射性耐光NADH类似物进行研究， 因为实验已经表明对这种物质的特异性吸收 化合物。一系列新的叶酸荧光类似物将被 在上述酶结构比较中， 这些衍生物也将被用作蝶啶和蝶呤的探针。 叶酸细胞转运及其作为细胞耐药标志物的研究 与DHFR升高引起的抗叶酸有关。底物的类似物 胸苷合成酶和磷酸核糖基甘氨酰胺(GAR) 甲酰基转移酶将被合成为两种探针 酶的作用机制及其协同性的测定 已建立的抗叶酸的化疗潜力 代谢物。此外，使用不同替换的 四氢蝶啶类化合物已在本实验室合成 氧化实验将作为模型进行 酶促芳香氨基酸羟基化反应， 需要减少的蝶呤作为底物。

As part of an established program in pteridine/folate chemistry and enzymology, this proposal is focused on two areas, the comparative structure and mechanism of rat liver dihydropteridine and dihydrofolate reductases (DHPR and DHFR) and the chemistry of folate/pteridine analogs that could act as mechanistic probes or inhibitors of enzymes in folate/pteridine metabolism, and might also possess chemotherapeutic potential. The two reductases are important because they participate in metabolic cycles (purine, pyrimidine and catecholamine biosyntheses) crucial to life. DHPR has been obtained crystalline, heavy atom derivatives have been made and preliminary X-ray crystallographic measurements carried out as a prelude to obtaining the complete three dimensional structure. To help achieve this goal the enzyme has been cloned and sequenced. During the next project period it is intended to complete the X-ray work and make definitive comparisons with the known mammalian DHFR structures to relate binding sites, homology, possible evolutionary divergence and comparative reaction mechanisms. To assist in the achievement of these goals the DHPR gene is to be expressed in Escherichia coli and site specific mutations will be introduced to determine the ligand-host interactions of key amino acids at the enzyme binding sites. The information will be used to predict pterin structural analogs which might strongly inhibit DHPR, since as yet no tight binding inhibitor is known for this enzyme. Such a molecule would be invaluable for examining DHPR metabolism and possibly help discover reasons for its ubiquitous tissue distribution since its cooperative action with aromatic amino acid hydroxylases is apparent only in liver, kidney, and nerve tissue. To complement the overall structural analysis of DHPR the nucleotide binding site is to be investigated using radioactive photolabile NADH analogs, as experiments have already indicated specific uptake for such compounds. A series of novel fluorescent folate analogs are to be synthesized for use.in the above enzyme structural comparisons, these.derivatives will also be used as probes for pteridine and folate cellular transport and as markers for cellular resistance to antifolates caused by elevated DHFR. Analogs of substrates for thymidylate synthase and phosphoribosylglycinamide (GAR) formyltransferase are to be synthesi both as probes of the enzymatic mechanisms and for the determination of their cooperative chemotherapeutic potential with the established antifolate metabolites. Additionally, using variously substituted tetrahydropteridines previously synthesi in this laboratory oxidative experiments are to be carried out as models for the enzymatic aromatic amino acid hydroxylation reactions, which require reduced pterins as substrates.