STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

二氢叶酸还原酶的结构研究

• 批准号: 3170027
• 负责人: RAYMOND L BLAKLEY
• 金额: $ 12.42万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-09-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3170027
• 关键词: DNA; Escherichia coli; NAD(H) phosphate; chemical binding; chemical structure function; circular dichroism; conformation; dihydrofolate reductase; electron spin resonance spectroscopy; enzyme structure; enzyme substrate; enzyme substrate complex; fluorescence spectrometry; folate; human subject; human tissue; isomer; leukemia; liver; lymphoblast; lymphocytic leukemia; methotrexate; neoplasm /cancer chemotherapy; neoplasm /cancer relapse /recurrence; neoplastic cell; nuclear magnetic resonance spectroscopy; pediatric neoplasm /cancer; point mutation; polyglutamates; stop flow technique; tissue /cell culture

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.

目的是研究二氢叶酸酶的结构