Mechanistic Studies of Lipoic Acid Synthase

描述（由申请人提供）：硫辛酸是一种必需的 参与的几种多酶复合物的含硫辅因子 在初级代谢中。多年来，人们一直认为， 直接从辛酸，但硫插入的机制， 未活化的脂肪酸仍然不清楚。最近，一个基因被克隆出来 这似乎是酶活性的催化核心， 参与了硫插入反应。在其推断的签名图案 蛋白质的一级序列和初始光谱表征 共同表明，这种“硫辛酸合成酶”属于一种新兴的 一类含有铁硫簇的酶， S-腺苷-L-甲硫氨酸作为产生碳中心自由基的手段 酶促反应的中间体。S-腺苷-L-甲硫氨酸和 这类酶中的铁-硫簇代表了对 生物化学教科书中传统上赋予它们的角色。 铁硫团簇传统上被认为是电子转移 试剂或刘易斯酸催化剂，而S-腺苷-L-甲硫氨酸仍然是 被认为主要是细胞甲基化剂。提出的实验 本文试图揭示与这些相关的“新”分子酶学， 辅因子几个实验机制进行了讨论，实验是 这将使他们之间的区别。 除了硫辛酸作为辅因子参与酶复合物外， 能量代谢，已知其调节患有糖尿病的患者中的葡萄糖代谢。 II型糖尿病和作为一个一般的细胞抗氧化剂，其中许多 其他的事有明显的证据表明，硫辛酸可以 在哺乳动物细胞中内源性合成。在此概述的实验 建议将奠定基础，研究是否 不能内源性合成硫辛酸可导致 细胞功能

DESCRIPTION (provided by applicant): Lipoic acid is an essential sulfur-containing cofactor of several multienzyme complexes that are involved in primary metabolism. It has been known for a number of years to be derived directly from octanoic acid, but the mechanism of sulfur insertion into the unactivated fatty acid has remained obscure. Recently, a gene has been cloned which appears to be the catalytic core of the enzymatic activity that is involved in the sulfur insertion reaction. Signature motifs within its deduced primary sequence and initial spectroscopic characterization of the protein collectively indicate that this "lipoic acid synthase" belongs to an emerging class of enzymes that contain iron-sulfur clusters, and that use S-adenosyl-L-methionine as a means of generating carbon-centered radical intermediates in enzymatic reactions. The roles of S-adenosyl-L-methionine and iron-sulfur clusters in this class of enzymes represent departures from the roles that they have traditionally been assigned in biochemical textbooks. Iron-sulfur clusters have traditionally been thought of as electron transfer agents or Lewis acid catalysts, while S-adenosyl-L-methionine is still considered to be mainly a cellular methylating agent. The experiments proposed herein seek to reveal the "new" molecular enzymology associated with these cofactors. Several experimental mechanisms are discussed, and experiments are proposed that will distinguish among them. Aside from the involvement of lipoic acid as a cofactor in enzyme complexes of energy metabolism, it is known to modulate glucose metabolism in patients with type II diabetes and to serve as a general cellular antioxidant, among many other things. There is significant evidence that lipoic acid can be endogenously synthesized in mammalian cells. Experiments outlined in this proposal will lay a foundation upon which studies to address whether the inability to synthesize lipoic acid endogenously can lead to a compromising of cellular function.