STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

目的是研究二氢叶酸酶的结构 还原酶（DHFR），这是治疗中使用的抗叶酸剂的靶点 癌症（例如，甲氨蝶呤、MTX;三甲蝶呤等），细菌 感染（甲氧苄啶）和寄生虫感染（乙胺嘧啶）。 多 拟议工作的一部分是关于如何研究的结构要素， 酶活性位点与抑制剂相互作用，从而导致 对后者的约束力非常强。 停流分光光度法和 将使用荧光测定法继续测量以下物质的速率常数： 与来自屎链球菌的DHFR结合和从其释放的抑制剂（两种 同工酶，SFDHFR 1和2），干酪乳杆菌，大肠杆菌，牛 肝脏、鸡肝和WIL 2人淋巴母细胞。 这些调查 还将提供有关初始抑制剂异构化的信息 涉及构象变化的复合物， 约束力 关于这些异构化的信息也将通过 研究抑制的发展动力学和循环的变化， 多色性 叶酸盐、二氢叶酸盐和5-去氮唑酸盐的结合， 将类似地研究它们的络合物的异构化。 效果 核苷酸（NADPH或其类似物）的存在， 还将研究这些过程中的地点。 的其他信息 结构特征和配体结合的相互关系， 复合物的构象变化将通过产生 野生型DHFR。 突变体E.杆菌 用Asn或Ser取代活性位点Asp的DHFR是可用的 并将用于上述研究。 SFDHFR 1和2的突变体将被 构建并用于识别影响催化活性的结构特征 效率和底物特异性。 其他变种人将被用来 增强对13C谱的解释。 13C标记的叶酸和抑制剂 还将对这些报告进行综合， 催化和抑制剂结合。 还将搜索 MTX治疗复发的肿瘤患者中的变异DHFR。

The objective is to study the structure of the enzyme dihydrofolate reductase (DHFR) which is the target for antifolates used in the treatment of cancer (e.g., methotrexate, MTX; trimetrexate; etc.), of bacterial infections (trimethoprim) and parasitic infections (pyrimethamine). Much of the proposed work is related to study of how the structural elements of the enzyme active site interact with the inhibitors, so as to result in very tight binding of the latter. Stopped-flow spectrophotometry and fluorimetry will be used to continue measurements of rate constants for inhibitor binding to and release from DHFR from Streptococcus faecium (two isoenzymes, SFDHFR 1 and 2), Lactobacillus casei, Escherichia coli, bovine liver, chicken liver and WIL2 human lymphoblasts. These investigations will also provide information on isomerizations of the initial inhibitor complexes involving conformational changes that are critical in increasing binding. Information on these isomerizations will also be obtained by studying the kinetics of development of inhibition and changes in circular doichroism. Binding of folate, dihydrofolate and 5-deazafolate and isomerizations of their complexes will be similarly studied. The effect that the presence of nucleotide (NADPH or its analogs) in the catalytic site on these processes will also be studied. Additional information on the interrelation of structural features and ligand binding and conformational changes of complexes will be sought by creating mutants of wild-type DHFR by oligonucleotide-directed mutagenesis. Mutants of E. coli DHFR with replacement of the active site Asp by Asn or Ser are available and will be used in the above studies. Mutants of SFDHFR 1 and 2 will be constructed and used to identify structural features affecting catalytic efficiency and substrate specificity. Other mutants will be used to enhance interpretation of 13C spectra. 13C labeled folates and inhibitors will also be synthesized to provide information on the mechanism of catalysis and of inhibitor binding. There will also be a search for variant DHFR in tumors of patients who relapse on MTX therapy.