Irsch Thorsten, Krauth-Siegel R Luise
Biochemie-Zentrum der Universität Heidelberg, 69120 Heidelberg, Germany.
J Biol Chem. 2004 May 21;279(21):22209-17. doi: 10.1074/jbc.M401240200. Epub 2004 Feb 19.
The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with k(cat)/K(m) values of 5 x 10(5) m(-1) s(-1) and 7 x 10(5) m(-1) s(-1), respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids.
乙二醛酶系统是一条普遍存在的途径，催化谷胱甘肽依赖的酮醛解毒反应，如甲基乙二醛，它主要作为糖酵解的副产物形成。编码乙二醛酶II的基因已从非洲昏睡病的病原体布氏锥虫中克隆出来。推导的蛋白质序列包含高度保守的金属结合基序THXHXDH，但缺少在人乙二醛酶II晶体结构中显示用于固定谷胱甘肽硫酯底物的三个碱性残基。重组布氏锥虫乙二醛酶II能水解乳酰谷胱甘肽，但对于乙二醛酶II的经典底物，在高达5 mM时未表现出饱和动力学。相反，寄生虫酶强烈偏好锥虫硫醇（双（谷胱甘肽基）亚精胺）的硫酯，它们由甲基乙二醛和锥虫硫醇制备，并通过高效液相色谱和质谱分析。单（乳酰）锥虫硫醇和双（乳酰）锥虫硫醇被布氏锥虫乙二醛酶II水解，催化常数与米氏常数的比值（k(cat)/K(m)）分别为5×10⁵ m⁻¹ s⁻¹和7×10⁵ m⁻¹ s⁻¹，产生d-乳酸并使锥虫硫醇再生。乙二醛酶II存在于布氏锥虫的哺乳动物血流形式和昆虫前循环形式中，是迄今已鉴定的动质体目首个乙二醛酶II。我们的结果表明，乙二醛酶系统是另一条途径，在锥虫中，几乎普遍存在的谷胱甘肽被独特的锥虫硫醇所取代。
