CHEMISTRY OF FOLATE AND PTERIDINE COENZYMES

作为蝶啶/叶酸化学既定计划的一部分， 酶学，这个建议是集中在两个领域，比较 大鼠肝脏二氢蝶啶结构与作用机制 二氢叶酸还原酶（DHPR和DHFR）和 叶酸/蝶啶类似物，其可以作为机械探针， 叶酸/蝶啶代谢酶的抑制剂， 也具有化疗潜力。 这两种还原酶是 重要是因为它们参与代谢循环（嘌呤， 嘧啶和儿茶酚胺生物合成）对生命至关重要。 DHPR 已获得结晶，重原子衍生物已 进行了初步的X射线晶体学测量 作为一个前奏，以获得完整的三维 结构 为了帮助实现这一目标， 和序列测定 在下一个项目期间， 完成X射线工作，并与 已知的哺乳动物DHFR结构涉及结合位点，同源性， 可能的进化分歧和比较反应 机制等 为了帮助实现这些目标，人权和人民代表部 基因将在大肠杆菌中表达，并具有位点特异性 将引入突变以确定配体-宿主 关键氨基酸在酶结合位点的相互作用。 的 信息将用于预测蝶呤结构类似物， 可能强烈抑制DHPR，因为迄今为止还没有紧密结合 已知该酶抑制剂。 这样的分子 对检查DHPR代谢非常有价值，可能有助于发现 其无处不在的组织分布的原因，因为它 与芳香族氨基酸羟化酶的协同作用， 仅在肝脏肾脏和神经组织中可见。 以补充 DHPR的核苷酸结合位点的整体结构分析 将使用放射性光不稳定的NADH类似物进行研究， 因为实验已经表明了这种特定的吸收， 化合物. 一系列新的荧光叶酸类似物将被 为use.in合成上述酶结构比较， 这些衍生物也可用作蝶啶的探针， 叶酸细胞转运和作为细胞抗性的标记 去氢叶酸还原酶升高引起的抗叶酸剂 基质类似物， 胸苷酸合成酶和磷酸核糖甘氨酰胺 甲酰基转移酶的合成都作为探针的 酶的机制和确定它们的合作 与已确定的抗叶酸剂的化疗潜力 代谢物。 此外，使用各种取代的 四氢蝶啶以前在这个实验室合成 氧化实验将作为模型进行， 酶促芳香族氨基酸羟基化反应， 需要还原蝶呤作为底物。

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.