STUDIES OF TRIMETHYLAMINE DEHYDROGENASE

描述：（改编自申请人的摘要） 拟议的研究是为了更深入地了解 三甲基胺脱氢酶（TMADH）是一组重要的酶 其含有多个氧化还原活性中心， 涉及这些位点之间的电子转移的机制。除了 它们的生理意义，这些蛋白质作为非常有用的 系统中检查的因素，支配磁和 这些中心之间的电子转移相互作用。催化的反应 TMADH，氧化脱甲基的三甲基胺二甲胺和 甲醛，是相当大的内在利益，了解 生物系统中的异咯嗪环可用的化学。这 化学与许多其他酶有关，包括临床上的 重要的酶如单胺氧化酶。 拟议的工作建立在PI以前的工作基础上，并具有以下内容 主要目的：检测特定氨基酸残基的作用 在TMADH催化的反应中; TMADH反应的表征 使用时间分辨晶体学;表征异常强的 TMADH的黄素和Fe_4S_4半醌中心之间的磁性相互作用; 的电化学和电子转移性能的检查 以及三甲胺与三甲基胺之间相互作用的表征 脱氢酶及其生理电子受体，和 电子转移黄素蛋白以三维结构为导向 的三甲胺脱氢酶，特定的氨基酸残基的作用， 酶在这些区域中的每一个的活性位点将是 通过定点诱变来解决。这项工作的目的是 全面了解结构与功能的关系 在三甲基胺脱氢酶中，这进一步加深了我们对 这一机制和相关酶。

DESCRIPTION: (adapted from applicant's abstract) The overall goal of the proposed research is to gain deeper insight into the mechanism of action of trimethylamine dehydrogenase (TMADH), a member of an important group of enzymes that contain multiple redox-active centers and which have overall catalytic mechanisms involving electron transfer between these sites. In addition to their physiological significance, these proteins serve as extremely useful systems in which to examine the factors which govern the magnetic and electron-transfer interactions between such centers. The reaction catalyzed by TMADH, the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde, is of considerable intrinsic interest in understanding the chemistry available to the isoalloxazine ring in biological systems. This chemistry is relevant to a number of other enzymes, including such clinically important enzymes as monoamine oxidase. The proposed work builds on previous work by the PI and has the following principal objectives: Examination of the role of specific amino acid residues in the reaction catalyzed by TMADH; characterization of the reaction of TMADH using time-resolved crystallography; characterization of the unusually strong magnetic interaction between the flavin and Fe4S4 semiquinone centers of TMADH; examination of the electrochemical and electron-transferring properties of TMADH; and characterization of the interaction between trimethylamine dehydrogenase and its physiological electron acceptor, and electron-transferring flavoprotein. Guided by the three-dimensional structure of trimethylamine dehydrogenase, the roles of specific amino acid residues in the active site of the enzyme with regard to each of these areas will be addressed by site-directed mutagenesis. The intention of this work is to construct a comprehensive picture of the relationship of structure to function in trimethylamine dehydrogenase that furthers our basic understanding of the mechanism of this and related enzymes.