Department of Biochemistry and the Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588, USA.
J Biol Chem. 2010 May 7;285(19):14459-66. doi: 10.1074/jbc.M110.104802. Epub 2010 Mar 10.
Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A; R = 19.9%, R(free) = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site and disrupts the previously observed Mg(2+) coordination in the ATP-binding site. l-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 A; R = 18.1%, R(free) = 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.
谷氨酰胺半胱氨酸连接酶（GCL）的结构特征，该酶催化谷胱甘肽生物合成的初始限速步骤，揭示了许多底物识别的分子细节。为了进一步描绘这个关键酶的机制细节，我们已经确定了两种抑制形式的酿酒酵母 GCL（ScGCL）的结构，该酶与人酶具有显著的序列同一性。在体内，GCL 活性受谷胱甘肽反馈调节。ScGCL-谷胱甘肽复合物结构的研究（2.5 A；R = 19.9%，R(free) = 25.1%）表明，抑制剂占据了谷氨酸和假定的半胱氨酸结合位点，并破坏了先前在 ATP 结合位点观察到的 Mg2+配位。l-丁硫氨酸-S-亚砜亚胺（BSO）是 GCL 的一种基于机制的抑制剂，已广泛用于细胞培养和体内模型系统中谷胱甘肽的耗竭。ScGCL-BSO 结构的检查（2.2 A；R = 18.1%，R(free) = 23.9%）证实，BSO 在亚砜亚胺氮上被磷酸化生成抑制性物质，并揭示了可能有助于过渡态稳定的接触。总的来说，这些结构推进了我们对这种关键酶的分子调节的理解，并提供了该酶催化机制的更多细节。
