Mechanistic Studies of Lipoic Acid Synthase

说明(申请人提供)：硫辛酸是一种必需的含硫辅因子，存在于几种参与能量代谢的多酶复合体中。在其功能形式中，它共价连接到指定的硫辛基携带蛋白上的特定赖氨酸残基的epsilon氨基上。它在这些复合体中发挥作用的详细方式已经知道了很长一段时间，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，人们才开始对其生物合成有一个清晰的了解。硫辛酸是以辅因子形式而不是游离酸形式生物合成的，这需要两种蛋白质专门用于转化。第一，辛酰基转移酶(LipB)催化八碳脂肪酰链从辛酰基-ACP转移到细胞中的几种硫辛基携带蛋白之一。第二种蛋白质，硫辛基合成酶(LIPA)，催化在脂肪酰基的第6和第8位插入两个硫原子，提供硫酰辅因子。LiPA是自由基SAM超家族中的一员，它利用[4Fe-4S]和S-腺苷蛋氨酸生成高能自由基，这些自由基是每个反应的中间产物。这项提议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。将特别关注LIPA，以准确地了解插入到衬底中的硫原子是从哪里获得的。这些努力将涉及细菌遗传学、暂态动力学和光谱学(UV-Vis、EPR、穆斯堡尔谱、Endor和FT-ICR质谱学)。除了硫辛酸作为能量代谢酶复合体的辅助因子外，人们还知道硫辛酸调节II型糖尿病患者的葡萄糖代谢，并作为一般细胞抗氧化剂等。有重要证据表明，硫辛酸可以在哺乳动物细胞内合成，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.