Lueder D V, Phillips M A
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
J Biol Chem. 1996 Jul 19;271(29):17485-90. doi: 10.1074/jbc.271.29.17485.
The parasitic protozoan Trypanosoma brucei maintains redox balance by synthesizing a conjugate of glutathione and spermidine termed trypanothione. The first committed step in the biosynthesis of glutathione, and thereby trypanothione, is catalyzed by the enzyme gamma-glutamylcysteine synthetase (gammaGCS). We have cloned and sequenced the 2037-base pair gene coding for the catalytic subunit of T. brucei gammaGCS. T. brucei gammaGCS appears to be encoded by a single copy gene. A transcript of about 2.3 kilobases was observed in procyclic trypomastigotes. The deduced amino acid sequence of 679 amino acids shares 45, 41, and 36% sequence identity with mammalian, Caenorhabditis elegans, and yeast gammaGCS, respectively. The T. brucei gammaGCS gene was expressed in E. coli; the purified 77.4-kDa enzyme catalyzes the ligation of L-Glu to L-Cys with a kcat of 10 s-1, confirming that the gene encodes the functional catalytic subunit of gammaGCS. The apparent Km values measured for the three natural substrates L-Glu, L-Cys, and ATP are 0.24, 0.69, and 0.07 mM, respectively. Unlike the mammalian enzyme, L-alpha-aminobutyrate (apparent Km = 10 mM) is a poor substitute for L-Cys in the T. brucei gammaGCS-catalyzed reaction. T. brucei gammaGCS is feedback-inhibited by glutathione (apparent KI = 1.1 mM), and it is inactivated by cystamine and buthionine sulfoximine. The kinetic properties of recombinant T. brucei gammaGCS suggest that the substrate binding pocket and the mechanism of enzyme regulation differ from the mammalian enzyme, providing evidence that T. brucei gammaGCS could be a selective chemotherapeutic target for the treatment of trypanosomiasis.
寄生原生动物布氏锥虫通过合成一种名为锥虫硫醇的谷胱甘肽与亚精胺的共轭物来维持氧化还原平衡。谷胱甘肽生物合成的首个关键步骤，也就是锥虫硫醇的生物合成步骤，是由γ-谷氨酰半胱氨酸合成酶（γGCS）催化的。我们已经克隆并测序了编码布氏锥虫γGCS催化亚基的2037个碱基对的基因。布氏锥虫γGCS似乎由一个单拷贝基因编码。在原循环型锥鞭毛体中观察到了一个约2.3千碱基的转录本。推导得到的679个氨基酸的氨基酸序列与哺乳动物、秀丽隐杆线虫和酵母的γGCS分别具有45%、41%和36%的序列同一性。布氏锥虫γGCS基因在大肠杆菌中表达；纯化后的77.4 kDa酶催化L-Glu与L-Cys的连接，催化常数为10 s-1，证实该基因编码γGCS的功能性催化亚基。对三种天然底物L-Glu、L-Cys和ATP测得的表观Km值分别为0.24、0.69和0.07 mM。与哺乳动物酶不同，在布氏锥虫γGCS催化的反应中，L-α-氨基丁酸（表观Km = 10 mM）是L-Cys的不良替代物。布氏锥虫γGCS受到谷胱甘肽的反馈抑制（表观KI = 1.1 mM），并且被胱胺和丁硫氨酸亚砜胺灭活。重组布氏锥虫γGCS的动力学特性表明，其底物结合口袋和酶调节机制与哺乳动物酶不同，这为布氏锥虫γGCS可能成为治疗锥虫病的选择性化疗靶点提供了证据。
