METHANE MONOOXYGENASE STRUCTURE AND MECHANISM

甲烷单加氧酶的结构和机制

• 批准号: 3298028
• 负责人: JOHN D LIPSCOMB
• 金额: $ 11.91万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298028
• 关键词: Methanobacteriaceae; Mossbauer spectrometry; Raman spectrometry; X ray spectrometry; catalyst; cofactor; electron spin resonance spectroscopy; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; flavoproteins; freezing; iron; ligands; metalloenzyme; methane; methanol; microorganism metabolism; nuclear magnetic resonance spectroscopy; oxygen; oxygenases; stop flow technique

We propose to investigate the active site structure and the molecular mechanism of catalysis by Methane Monooxygenase (MMO) purified from the Type II methanotroph, Methylosinus trichosporium OB3b (M.t. OB3b). This enzyme catalyzes the first step in the bacterial oxidation of methane (producing methanol) and will adventitiously catalyze hydroxylation of many other saturated and unsaturated hydrocarbons. The mechanism of this 3 component enzyme system is unknown. However, studies with this enzyme as well as MMOs isolated from 2 other sources suggest that the component which actually catalyzes the hydroxylation contains none of the cofactors known to exist in other monooxygenases. Thus MMO probably uses a new mechanism for catalysis. We have purified all of the components of M.t. OB3b MMO. The system offers significant advantages over the 2 other purified MMO systems including greater stability, greater yield and a 15-fold increase in specific activity. These properties will allow the purification of gram quantities of enzyme so that biophysical techniques including optical, EPR, Mossbauer and EXAFS spectroscopies can be applied to determine the structure of the hydroxylase active site. Spectroscopy of small ligand complexes, mechanism based inhibitors, isotopically labeled substrates and inhibitors, and transient kinetics are proposed as methods to investigate the molecular mechanism. This work should yield a fundamental understanding of a new type of oxygen activation chemistry, a new role for Fe in this chemistry and perhaps new insight into the design of catalysts for oxidation of abundant hydrocarbons.

我们建议研究主动地点结构和 甲烷单加氧酶催化的分子机制 （MMO）从II型甲烷营养物，甲基素纯化 Trichosporium ob3b（M.T。OB3b）。 这种酶催化了第一个 进入甲烷（产生甲醇）的细菌氧化 并将异议地催化许多其他的羟基化 饱和和不饱和烃。 这个3的机制 组件酶系统尚不清楚。 但是，研究 这种酶以及从其他2个来源分离的MMO 建议实际上催化的组件 羟基化不包含已知存在的辅助因子 其他单加氧酶。 因此，MMO可能使用了新的 催化机制。 我们已经纯化了所有组件 M.T. OB3B MMO。 该系统提供了很大的优势 在其他两个纯化的MMO系统中，包括更大的稳定性， 特定活动的产量更高，增加了15倍。 这些 属性将允许纯化量的数量 酶使包括光学，EPR在内的生物物理技术 可以应用MOSSBAUER和EXAFS光谱镜来确定 羟化酶活性位点的结构。 光谱 小配体配合物，基于机制的抑制剂，同位素 标记的底物和抑制剂以及瞬态动力学是 提出作为研究分子机制的方法。 这项工作应该对一种新类型产生基本理解 氧气激活化学，Fe在此中的新作用 化学，也许是对催化剂设计的新见解 丰富的碳氢化合物的氧化。