Koenig K, Menge U, Kiess M, Wray V, Flohé L
Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
J Biol Chem. 1997 May 2;272(18):11908-15. doi: 10.1074/jbc.272.18.11908.
Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculata by aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 micromol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting Km values for Mg2+-ATP, GSH, and spermidine of 0.25 +/- 0.02, 2.51 +/- 0.33, and 0.47 +/- 0. 09 mM, respectively, and a kcat of 415 +/- 78 min-1. Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (Ki = 0. 08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (Ki = 0.48 mM).
锥虫硫醇是锥虫氧化防御系统中的必需代谢物，由两种不同的蛋白质谷胱甘肽亚精胺合成酶和锥虫硫醇合成酶合成。通过双水相系统和色谱法从锥虫纤细短膜虫中纯化谷胱甘肽亚精胺合成酶至均一。该酶的比活性为每毫克蛋白质每分钟形成38微摩尔谷胱甘肽亚精胺。在SDS聚丙烯酰胺凝胶电泳中其分子量为78 kDa，在天然聚丙烯酰胺凝胶电泳和凝胶过滤中主要呈单体形式。等电点为pH 4.6，最适pH接近7.6。部分氨基酸测序显示与大肠杆菌的谷胱甘肽亚精胺合成酶/酰胺酶有同源性，但相似性较低，在纤细短膜虫谷胱甘肽亚精胺合成酶中未检测到酰胺酶活性。锥虫谷胱甘肽亚精胺合成酶的动力学显示为快速平衡随机机制，Mg2+-ATP、GSH和亚精胺的极限Km值分别为0.25±0.02、2.51±0.33和0.47±0.09 mM，kcat为415±78 min-1。在限制共底物供应的情况下，31P NMR未检测到部分反应，这支持了催化需要形成四聚体复合物的观点。ADP对ATP具有竞争性抑制作用（Ki = 0.08 mM），锥虫硫醇对GSH具有竞争性反馈抑制作用（Ki = 0.48 mM）。
