INVESTIGATIONS INTO KINETICS OF ENZYMES VIA NMR

通过 NMR 研究酶的动力学

• 批准号: 6120980
• 负责人: DEXTER B NORTHROP
• 金额: $ 0.04万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120980
• 关键词: biological products; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; proteins

Toluene 4-monooxygenase (T4MO) catalyzes NADH- and O2-dependent conversion of toluene to p-cresol, and can also catalyze the adventitious oxidation of numerous other hydrocarbons such as indole, naphthalene, chloroform, and trichloroethylene. This four-protein complex consists of an NADH oxidoreductase, a catalytic effector protein, a diiron center-containing hydroxylase, and a small Mr soluble ferredoxin (T4MOC, 12,326Da). T4MOC is a monomeric protein which contains a Rieske-type iron sulfur center. Catalytic reconstitution studies have shown that T4MOC is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components. We will conduct research to determine 1D, 2D and 3D structures of T4MOC by using multinuclear high field NMR and isotopically labeled protein samples. These studies will provide electronic and dynamic information about correlations between redox states, proton exchange, and spin delocalization that may contribute to electron transfer reactions between Riseke and diiron centers. Moreover, by using the completely soluable T4MO enzyme system, we will study changes in T4MOC redox and protonation states, coordination geometry, and electron delocalization upon complex formation with T4MOH, in catalytically relevant electron transfer complexes.

甲苯4-单加氧酶(T4MO)催化NADH和O2依赖 将甲苯转化为对甲酚，还可以催化 许多其他碳氢化合物的不定氧化，如吲哚， 萘、氯仿和三氯乙烯。这四种蛋白质 复合体由NADH氧化还原酶，一种催化效应器组成 蛋白质，一个含有双铁中心的羟基酶和一个小的MR 可溶性铁氧还蛋白(T4MOC，12,326 Da)。T4moc是一种单体蛋白质 它包含一个Rieske类型的铁硫中心。催化作用 重建研究表明，T4MOC是强制性的 用于氧化还原酶和双铁之间的电子转移 羟基酶成分。我们将进行研究，以确定一维、二维 用多核高场核磁共振和核磁共振研究T4MOC的三维结构 同位素标记的蛋白质样本。这些研究将提供 关于氧化还原之间相互关系的电子和动态信息 态、质子交换和自旋离域 Riseke和DiIron中心之间的电子转移反应。 此外，通过使用完全可溶的T4MO酶系统，我们将 研究T4MOC氧化还原和质子化状态的变化，配位 几何构型和形成络合物时电子离域 T4MOH，在催化相关的电子转移络合物中。