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助因子，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，它共价地连接到指定的携带脂酰的蛋白质上的特定赖氨酸残基的epsilon氨基。它在这些复合体中起作用的详细方式早已为人所知，几乎在每一本初级生物化学教科书中都能找到。相比之下，直到最近几年才对其生物合成有了清晰的认识。硫辛酸以其辅助因子形式而不是其游离酸形式进行生物合成，需要两种专门用于转化的蛋白质。首先，辛烷酰转移酶（LipB）催化八碳脂肪酰基链从辛烷酰acp转移到细胞中几种携带脂酰的蛋白质之一。第二种蛋白质，脂酰合成酶（LipA）催化两个硫原子在脂肪酰基的第6和第8位插入，形成脂酰辅助因子。LipA是自由基SAM超家族的一员，它利用a [4Fe-4S]和s -腺苷蛋氨酸产生高能自由基，这些自由基是每次反应的中间产物。这项提议的长期目标是在分子水平上详细了解这些酶是如何工作的。特别的焦点将指向LipA，以准确地了解插入底物的硫原子是从哪里衍生出来的。工作将涉及细菌遗传学，瞬态动力学和光谱学（UV-vis， EPR, M ' ssbauer， ENDOR和FT-ICR质谱）。除了硫辛酸作为辅助因子参与能量代谢的酶复合体外，它还可以调节II型糖尿病患者的葡萄糖代谢，并作为一种一般的细胞抗氧化剂，以及其他许多作用。有重要证据表明，硫辛酸可以在哺乳动物细胞中内源性合成，抑制这一途径会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.