Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding.
Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding.
复制标题
DOI：
10.1074/jbc.m107961200
复制
发表时间：
2002-01
期刊：
影响因子：
--
通讯作者：
Brenda S. Kelly;W. Antholine;O. Griffith
中科院分区：
文献类型：
--
作者：
Brenda S. Kelly;W. Antholine;O. Griffith
γ-谷氨酰半胱氨酸合成酶（γ-GCS，谷氨酸-半胱氨酸连接酶）催化谷胱甘肽生物合成的第一步和限速步骤，存在于许多原核生物和几乎所有真核生物中。尽管迄今为止检查的所有真核γ-GCS亚型均被丁硫氨酸亚磺酰亚胺（BSO）快速抑制，但大多数报告表明细菌γ-GCS对BSO具有抗性。我们在标准测定条件下用大肠杆菌 gamma-GCS 证实了后一个发现，表明与哺乳动物亚型相比，BSO 的初始结合亲和力降低，BSO 介导的失活率降低。我们还发现，在测定混合物中用 Mn2+ 替代 Mg2+ 会增加 BSO 的初始结合亲和力以及 BSO 导致基于机制的失活的速率。类似地，在 Mn2+ 存在下，大肠杆菌 gamma-GCS 对其氨基酸底物的特异性会变宽，并且对于一些非常差的底物的反应速率也会得到提高。这些结果表明二价金属离子在氨基酸与大肠杆菌γ-GCS 的结合中发挥作用。使用 Mn2+ 进行的电子顺磁共振 (EPR) 研究表明，大肠杆菌 gamma-GCS 结合两个二价金属离子； Mn2+ 的 Kd 值分别为 1.1 微米和 82 微米。 L-谷氨酸或 l-BSO 与两个 Mn2+/gamma-GCS 物质的结合产生了额外的上场和下场 X 波段 EPR 超细线，间隔为 45 G，结果表明两个 Mn2+ 是自旋耦合的，因此在活性位点中明显分开 5 A 或更小。其他 EPR 研究表明，Cu2+ 取代了 Mg2+ 或 Mn2+，表明 Cu2+ 在 γ-GCS 活性位点与 1 个 N 和 3 个 O 配体结合。在γ-GCS的催化机制及其与更充分表征的谷氨酰胺合成酶反应的关系的背景下讨论了结果。
Gamma-glutamylcysteine synthetase (gamma-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes. Although all eukaryotic gamma-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial gamma-GCS is resistant to BSO. We have confirmed the latter finding with Escherichia coli gamma-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms. We also find that substitution of Mn2+ for Mg2+ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation. Similarly, the specificity of E. coli gamma-GCS for its amino acid substrates is broadened in the presence of Mn2+, and the rate of reaction for some very poor substrates is improved. These results suggest that divalent metal ions have a role in amino acid binding to E. coli gamma-GCS. Electron paramagnetic resonance (EPR) studies carried out with Mn2+ show that E. coli gamma-GCS binds two divalent metal ions; Kd values for Mn2+ are 1.1 microm and 82 microm, respectively. Binding of l-glutamate or l-BSO to the two Mn2+/gamma-GCS species produces additional upfield and downfield X-band EPR hyperfine lines at 45 G intervals, a result indicating that the two Mn2+ are spin-coupled and thus apparently separated by 5 A or less in the active site. Additional EPR studies in which Cu2+ replaced Mg2+ or Mn2+ suggest that Cu2+ is bound by one N and three O ligands in the gamma-GCS active site. The results are discussed in the context of the catalytic mechanism of gamma-GCS and its relationship to the more fully characterized glutamine synthetase reaction.