Oza Sandra L, Chen Shoujun, Wyllie Susan, Coward James K, Fairlamb Alan H
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, UK.
FEBS J. 2008 Nov;275(21):5408-21. doi: 10.1111/j.1742-4658.2008.06670.x.
Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants K(GSH) = 609 microM, K(Spd) = 157 microM and K(ATP) = 215 microM. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (K(i) approximately 156 microM), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k(3) (on rate) = 6.98 x 10(4) M(-1) x s(-1) and k(4) (off rate) = 1.3 x 10(-3) s(-1), providing a dissociation constant K(i) = 18.6 nM. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with K(i)(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.
谷胱甘肽亚精胺是锥虫硫醇生物合成过程中形成的一种中间体，锥虫硫醇是动质体中抵御化学和氧化应激的一种必需代谢物。来自 fasciculata 克氏锥虫（CfGspS）的谷胱甘肽亚精胺合成酶（EC 6.3.1.8）的动力学机制遵循快速平衡随机双底物双产物模型，动力学常数为 K(GSH) = 609 μM、K(Spd) = 157 μM 和 K(ATP) = 215 μM。谷胱甘肽亚精胺的膦酸酯和次膦酸酯类似物，此前已证明是大肠杆菌 GspS 的有效抑制剂，对 CfGspS 同样有效。四面体膦酸酯作为谷胱甘肽（GSH）的简单基态类似物（K(i) 约为 156 μM），而次膦酸酯则表现为假定的不稳定四面体中间体的稳定模拟物。动力学研究表明，次膦酸酯表现为慢结合双底物抑制剂[对 GSH 和亚精胺（Spd）具有竞争性]，速率常数 k(3)（结合速率）= 6.98 × 10⁴ M⁻¹ × s⁻¹ 和 k(4)（解离速率）= 1.3 × 10⁻³ s⁻¹，解离常数 K(i) = 18.6 nM。次膦酸酯类似物还抑制了来自 fasciculata 克氏锥虫、硕大利什曼原虫、克氏锥虫和布氏锥虫的重组锥虫硫醇合成酶（EC 6.3.1.9），其 K(i)(app) 值比 CfGspS 大 20 - 40 倍。这种次膦酸酯类似物仍然是迄今为止鉴定出的最有效的酶抑制剂，是用于治疗锥虫病和利什曼病药物研发的良好起点。
