Brekken D L, Phillips M A
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
J Biol Chem. 1998 Oct 9;273(41):26317-22. doi: 10.1074/jbc.273.41.26317.
The parasitic protozoan Trypanosoma brucei utilizes a conjugate of glutathione and spermidine, termed trypanothione, in place of glutathione to maintain cellular redox balance. The first committed step in the biosynthesis of glutathione and thereby trypanothione, is catalyzed by gamma-glutamylcysteine synthetase (gamma-GCS). We have determined the kinetic mechanism for T. brucei gamma-GCS. The kinetics are best described by a rapid equilibrium random ter-reactant mechanism, in which the model derived Kd values for the binding of L-Glu, L-alpha-aminobutyrate, and ATP to free enzyme are 2.6, 5.1, and 1.4 mM, respectively. However, significant dependences exist between the binding of some of the substrate pairs. The binding of either ATP or L-Glu to the enzyme increases the binding affinity of the other by 18-fold, whereas the binding of L-Glu or L-alpha-aminobutyrate decreases the binding affinity of the other by 6-fold. Similarly to the mammalian enzyme, cystamine is a time-dependent, irreversible inhibitor of T. brucei gamma-GCS. It has been suggested by several studies that cystamine labels an active site Cys residue essential for catalysis. Among the enzymes reported to be inactivated by cystamine, only one Cys residue is invariant (Cys-319 in T. brucei gamma-GCS). Mutation of Cys-319 to Ala in T. brucei gamma-GCS renders the enzyme insensitive to cystamine inactivation without significantly affecting the enzyme's catalytic efficiency, kinetic mechanism, or substrate affinities. These studies suggest that cystamine inactivates the enzyme by blocking substrate access to the active site and not by labeling an essential active site residue.
寄生原生动物布氏锥虫利用谷胱甘肽与亚精胺的共轭物（称为锥虫硫醇）来替代谷胱甘肽，以维持细胞的氧化还原平衡。谷胱甘肽生物合成进而也是锥虫硫醇生物合成中的首个关键步骤，由γ-谷氨酰半胱氨酸合成酶（γ-GCS）催化。我们已确定了布氏锥虫γ-GCS的动力学机制。该动力学最好用快速平衡随机三反应物机制来描述，其中推导得出的L-谷氨酸、L-α-氨基丁酸和ATP与游离酶结合的Kd值分别为2.6、5.1和1.4 mM。然而，一些底物对之间存在显著的依赖性。ATP或L-谷氨酸与酶的结合会使另一种底物的结合亲和力增加18倍，而L-谷氨酸或L-α-氨基丁酸的结合会使另一种底物的结合亲和力降低6倍。与哺乳动物的酶类似，胱胺是布氏锥虫γ-GCS的一种时间依赖性不可逆抑制剂。多项研究表明，胱胺标记了催化所必需的活性位点半胱氨酸残基。在据报道被胱胺灭活的酶中，只有一个半胱氨酸残基是不变的（布氏锥虫γ-GCS中的Cys-319）。将布氏锥虫γ-GCS中的Cys-319突变为丙氨酸会使该酶对胱胺灭活不敏感，而不会显著影响酶的催化效率、动力学机制或底物亲和力。这些研究表明，胱胺通过阻止底物进入活性位点而使酶失活，而非通过标记必需的活性位点残基。
