Mechanisms of Dihydroorotate Dehydrogenases

二氢乳清酸脱氢酶的机制

• 批准号: 6693790
• 负责人: BRUCE A PALFEY
• 金额: $ 18.88万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6693790
• 关键词: Raman spectrometry; acidity /alkalinity; active sites; anaerobiosis; chemical chain length; chemical kinetics; computer simulation; drug design /synthesis /production; enzyme mechanism; enzyme model; enzyme substrate complex; flavin mononucleotide; intermolecular interaction; isozymes; microcalorimetry; model design /development; molecular dynamics; molecular genetics; orotate; oxidation reduction reaction; oxidoreductase; protein structure function; quinones; site directed mutagenesis; thermodynamics; thiols

DESCRIPTION: (provided by applicant) Reaction mechanisms will be determined for the family 1A dihydroorotate dehydrogenase (DHOD) from Lactococcus lactis and Enterococcus faecalis, and the family 2 enzymes from Homo sapiens and Escherichia coli. DHOD, the only redox enzyme in pyrimidine biosynthesis, is an attractive target for drug design in the treatment of many diseases, including malaria, arthritis, and Pneumocystis infections in AIDS patients. The mechanism of reduction of the enzyme-bound FMN by dihydroorotate for the two enzymes will be determined under anaerobic conditions through stopped-flow kinetic analyses, including the use of single- and double deuterium substrate isotope effects, solvent isotope effects, and the pH dependence of the rate constants. Mutant L. lactis A and E. coli enzymes will be studied in order to determine the roles that active site residues have in the reaction. The mechanism of oxidation of reduced L. lactis DHOD A by fumarate will be elucidated in stopped-flow experiments that will determine the pH dependence of the reaction, and in double-mixing experiments, the deuterium isotope effects for the transfer of each hydrogen to fumarate. The mechanism(s) of oxidation by quinones of DHOD A and the E. coli enzyme will be determined in stopped-flow experiments utilizing a range of quinone substrates. The ubiquinone isoprenyl chain-length preference of the E. coli enzyme will be determined. Enzyme-ligand interactions will be probed with Raman spectroscopy.

描述：（由申请人提供）将确定反应机制 乳酸乳球菌和 肠球菌肠球菌和家族2酶来自Homo Sapiens和 大肠杆菌。 DHOD是嘧啶生物合成中唯一的氧化还原酶，是一种 药物设计的有吸引力的目标，用于治疗许多疾病，包括 艾滋病患者的疟疾，关节炎和肺炎胸膜感染。机制 通过二氢易原酸盐减少酶结合的FMN的两种酶将 通过停止流动动力学分析在厌氧条件下确定， 包括使用单氘和双氘底物同位素效应， 溶剂同位素效应以及速率常数的pH依赖性。突变L. 将研究乳酸A和大肠杆菌酶以确定角色 活性位点残基有反应。氧化机制 富马酸乳酸乳酸乳杆菌DHOD A的减少将在停止流中阐明 将决定反应的pH依赖性的实验，并在 双混合实验，氘同位素的转移 每种氢向富马酸盐。 DHOD A喹酮氧化的机理 大肠杆菌酶将在使用的止流实验中确定 一系列喹酮底物。泛氨基异丙烯基链长偏好 将确定大肠杆菌酶的。酶 - 配体相互作用将是 用拉曼光谱探测。