Vickers Tim J, Fairlamb Alan H
Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
J Biol Chem. 2004 Jun 25;279(26):27246-56. doi: 10.1074/jbc.M311039200. Epub 2004 Apr 8.
Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the alpha, beta, and gamma subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the gamma subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bgamma genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1Bgamma had no S-transferase activity, suggesting that eEF1Bgamma is unstable in the absence of the other subunits.
锥虫硫醇是动质体目所特有的一种硫醇，已被证明是其抗氧化防御的重要组成部分。然而，关于锥虫硫醇在异生物质代谢中的作用却知之甚少。在硕大利什曼原虫、婴儿利什曼原虫、塔氏利什曼原虫、布氏锥虫和 fasciculata 短膜虫的提取物中检测到了锥虫硫醇 S -转移酶活性，但克氏锥虫中未检测到。在这些寄生虫中均未检测到谷胱甘肽 S -转移酶活性。从 fasciculata 短膜虫中纯化出了锥虫硫醇 S -转移酶，结果表明它是一种由三个亚基组成的十六聚体复合物，相对分子质量为 650,000。这种酶复合物对硫醇锥虫硫醇和谷胱甘肽亚精胺具有特异性，并且在一系列谷胱甘肽 S -转移酶底物中仅使用 1 -氯 - 2,4 -二硝基苯。肽序列分析表明这三个组分是核糖体真核延伸因子 1B（eEF1B）的α、β和γ亚基。复合物的部分解离表明 S -转移酶活性与γ亚基相关。此外，发现汽巴克隆蓝是一种紧密结合抑制剂，活性蓝 4 是一种不可逆的时间依赖性抑制剂，它仅共价修饰γ亚基。在锥虫硫醇存在的情况下，活性蓝 4 的失活速率增加了 600 多倍，并且汽巴克隆蓝可保护该酶不被 1 -氯 - 2,4 -二硝基苯失活，这证实了这些染料与活性位点区域相互作用。从 fasciculata 短膜虫中克隆了两个 eEF1Bγ基因，但重组的 fasciculata 短膜虫 eEF1Bγ没有 S -转移酶活性，这表明在没有其他亚基的情况下 eEF1Bγ不稳定。
