METHANE MONOOXYGENASE STRUCTURE AND MECHANISM

我们建议研究活性中心的结构和 甲烷单加氧酶催化作用的分子机制 (MMO)从甲氧菌II型甲烷嗜甲菌中提纯 毛孢霉OB3b(M.t.OB3b)。这种酶催化第一个 甲烷细菌氧化的步骤(产生甲醇) 会意外地催化许多其他的羟基化 饱和和不饱和碳氢化合物。这3个字的作用机制 组成酶系统尚不清楚。然而，研究表明， 该酶以及从其他两个来源分离的MMOS 这表明，实际上催化 羟化不含已知存在于 其他单加氧酶。因此，MMO可能使用新的 催化机理。我们已经提纯了所有的成分 M.T.OB3b MMO.该系统提供了显著的优势 比其他2个纯化的MMO系统更稳定， 更高的产量和15倍的比活跃度。这些 属性将允许提纯克量的 酶使生物物理技术包括光学、EPR、 穆斯堡尔和EXAFS光谱可以用来确定 羟基酶活性部位的结构。光谱学 小配体络合物，基于机理的抑制剂，同位素 标记的底物和抑制剂，以及瞬时动力学 被认为是研究其分子机制的方法。 这项工作应该对一种新的类型有一个基本的理解 氧活化化学中，铁在这方面的新作用 化学，也许还有对催化剂设计的新见解 富含碳氢化合物的氧化。

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.