Mechanistic Studies of Lipoic Acid Synthase

描述（由申请人提供）：硫辛酸是一种必需的含硫辅因子，存在于参与能量代谢的几种多酶复合物中。在其功能形式中，它共价连接到指定的硫辛酰基携带蛋白上的特定赖氨酸残基的N-氨基上。它在这些复合物中发挥作用的详细方式已经知道了相当长的一段时间，并且几乎在每一本初级生物化学教科书中都可以找到。相比之下，只有在最近几年才开始出现对其生物合成的清晰理解。硫辛酸以其辅因子形式而不是其游离酸形式生物合成，需要两种专门用于转化的蛋白质。第一，辛酰基转移酶（LipB）催化八碳脂肪酰基链从辛酰基-ACP转移到细胞中的几种携带硫辛酰基的蛋白质之一。第二种蛋白质，硫辛酰合酶（利帕）催化在脂肪酰基的6位和8位插入两个硫原子，提供硫辛酰辅因子。利帕是自由基SAM超家族酶的成员，其使用[4Fe-4S]和S-腺苷甲硫氨酸产生高能自由基，所述高能自由基是每个反应中的中间体。这项提案的长期目标是在详细的分子水平上了解这些酶是如何工作的。特别关注的是利帕，以准确理解插入底物的硫原子的来源。研究工作将涉及细菌遗传学、瞬态动力学和光谱学（紫外-可见、EPR、穆斯堡尔、ENDOR和FT-ICR质谱法）。除了作为能量代谢的酶复合物中的辅因子的硫辛酸的参与之外，已知其调节患有II型糖尿病的患者中的葡萄糖代谢并且充当一般的细胞抗氧化剂，以及许多其它物质。有显著的证据表明，硫辛酸可以在哺乳动物细胞中内源性合成，并且该途径的抑制损害细胞功能并导致死亡。

DESCRIPTION (provided by applicant): Lipoic acid is an essential sulfur-containing cofacor that is found in several multienzyme complexes that are involved in energy metabolism. In its functional form it is covaleritly attached to the epsilon amino group of a specific lysine residue on a designated lipoyl carrying protein. The detailed manner in which it functions in these complexes has been known for quite some time, and is found in almost every beginning biochemistry textbook. By contrast, only within the last few years has a clear understanding of its biosynthesis begun to emerge. Lipoic acid is biosynthesized in its cofactor form rather than its free acid form, requiring two proteins that are dedicated to the transformation. The first, octanoyl transferase (LipB) catalyzes the transfer of the eight-carbon fatty acyl chain from octanoyl-ACP to one of the several lipoyl carrying proteins in the cell. The second protein, lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at positions 6 and 8 of the fatty acyl group, affording the lipoyl cofactor. LipA is a member of radical SAM superfamily of enzymes, which use a [4Fe-4S] and S-adenosylmethionine to generate high-energy radicals that are intermediates in each reaction. The long-term objective of this proposal is to understand at the detailed molecular level how each of these enzymes work. Particular focus will be directed at LipA, to understand exactly from where the sulfur atom that is inserted into the substrate is derived. Efforts will involve bacterial genetics, transient state kinetics, and spectroscopy (UV-vis, EPR, M"ssbauer, ENDOR, and FT-ICR mass spectrometry). 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, and that the inhibition of this pathway compromises cellular function and leads to death.