Function of conserved residues of human glutathione synthetase - Implications for the Atp-grasp enzymes
Function of conserved residues of human glutathione synthetase - Implications for the Atp-grasp enzymes
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DOI：
10.1074/jbc.m401334200
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发表时间：
2004-05-21
影响因子：
4.8
通讯作者：
Anderson, ME
中科院分区：
文献类型：
--
作者：
Dinescu, A;Cundari, TR;Anderson, ME
谷胱甘肽合成酶是一种酶，属于谷胱甘肽合成酶atp结合结构域超家族。它以atp依赖的方式催化γ -谷氨酰半胱氨酸和甘氨酸生物合成谷胱甘肽的第二步。谷胱甘肽合成酶已从多种生物来源纯化和测序；尽管如此，它的确切机制还没有被完全理解。应用多种结构比对方法，在结合位点鉴定出4个高度保守的人谷胱甘肽合成酶残基（Glu-144、Asn-146、Lys-305和Lys-364）。通过实验和计算的定点诱变研究了这些基因的功能。从已报道的人谷胱甘肽合成酶晶体结构出发，采用分子力学和分子动力学模拟技术获得了几种人谷胱甘肽合成酶突变体酶的三维坐标。与圆二色光谱一致，我们的结果显示，残基突变后酶的整体结构没有发生重大变化。然而，半经验计算表明，配体结合受到这些突变的影响。保守残基和配体之间的关键相互作用被检测到，并发现对酶活性至关重要。特别是带负电荷的Glu-144残基在催化中起主要作用。
Glutathione synthetase is an enzyme that belongs to the glutathione synthetase ATP-binding domain-like superfamily. It catalyzes the second step in the biosynthesis of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Glutathione synthetase has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. The function of these was studied by experimental and computational site-directed mutagenesis. The three-dimensional coordinates for several human glutathione synthetase mutant enzymes were obtained using molecular mechanics and molecular dynamics simulation techniques, starting from the reported crystal structure of human glutathione synthetase. Consistent with circular dichroism spectroscopy, our results showed no major changes to overall enzyme structure upon residue mutation. However, semiempirical calculations revealed that ligand binding is affected by these mutations. The key interactions between conserved residues and ligands were detected and found to be essential for enzymatic activity. Particularly, the negatively charged Glu-144 residue plays a major role in catalysis.