PECASE: Mechanistic Studies on Cyclopropane Fatty Acid Synthase

机构：宾夕法尼亚州立大学帕克斯-腺苷- l-蛋氨酸（AdoMet）是酶催化中最通用的细胞代谢物之一。直到最近，它主要被认为是一种细胞甲基化剂，因为它是广泛的生物化合物的甲基的主要来源，包括DNA、RNA、蛋白质、脂质、碳水化合物和各种小分子。AdoMet还参与了其他各种有趣的转换。在一类酶中，它与铁硫簇协同作用，产生酶结合自由基，这是某些酶促反应的中间产物。在本文描述的项目中，它作为亚甲基而不是甲基的供体，在大肠杆菌环丙烷脂肪酸合成酶催化的一个迷人的反应中起作用。第二底物是存在于磷脂的不饱和脂肪酸酰基链中的分离的和未活化的顺式烯烃。与甲基转移相反，在AdoMet的亚甲基转移的生化文献中很少有先例。反应的所有极性机制都可能涉及能量很高的中间体。此外，由于大多数细胞磷脂是磷脂双层的组成部分，其中它们的脂肪酸链与水环境隔离，因此尚不清楚这种酶如何催化两种不同溶解度的底物之间的反应。该项目的目标是利用各种动力学、机械和物理技术来解决这些有趣的问题，如果成功，将对酶反应机制的一般理解做出重大贡献。该项目最初是作为职业奖资助的，并于2004年5月转换为工程师和科学家的总统早期职业奖（PECASE）奖。

Proposal Title: PECASE: Mechanistic Studies on Cyclopropane Fatty Acid SynthaseInstitution: Pennsylvania State Univ University ParkS-adenosyl-L-methionine (AdoMet) is one of the most versatile cellular metabolites used in enzymatic catalysis. Until relatively recently, it has been appreciated primarily as a cellular methylating agent, since it is the primary source of methyl groups for a broad spectrum of biological compounds, including DNA, RNA, proteins, lipids, carbohydrates, and a diverse array of small molecules. AdoMet is also involved in a variety of other interesting transformations. In one class of enzymes, it functions in concert with iron-sulfur clusters to generate enzyme-bound radicals that are intermediates in certain enzymatic reactions. In the project described herein, it functions as a donor of a methylene group rather than a methyl group, in a fascinating reaction catalyzed by the enzyme cyclopropane fatty acid synthase from Escherichia coli. The second substrate is an isolated and unactivated cis olefin present in the unsaturated fatty acid acyl chains of phospholipids. In contrast to methyl transfer, there is very little precedent in the biochemical literature for methylene transfer from AdoMet. All polar mechanisms for the reaction would be expected to involve intermediates that are very high in energy. In addition, since most cellular phospholipids are constituents of phospholipid bilayers, wherein their fatty acid chains are sequestered from the aqueous milieu, it is unclear how this enzyme catalyzes a reaction between two substrates of opposing solubilities. The goal of this project is to address these intriguing questions using a variety of kinetic, mechanistic, and physical techniques, which if successful, will contribute significantly to the general understanding of enzyme reaction mechanisms.This project was originally funded as a CAREER award, and was converted to a Presidential Early Career Award for Engineers and Scientists (PECASE) award in May 2004.