Abbott Jared J, Ford Jennifer L, Phillips Margaret A
Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, 75390-9041, USA.
Biochemistry. 2002 Feb 26;41(8):2741-50. doi: 10.1021/bi0159128.
gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the ATP-dependent ligation of L-Glu and L-Cys, which is the first step in de novo biosynthesis of the tripeptide glutathione. Recently it was demonstrated that gamma-GCS is a structural homologue of glutamine synthetase (GS), providing the basis to build a model for the gamma-GCS active site [Abbott et al. (2001) J. Biol. Chem. 276, 42099-42107]. Substrate binding determinants in the active site of gamma-GCS have been identified and characterized in the enzyme from the parasitic protozoa Trypanosoma brucei using this model as a guide for site-directed mutagenesis. R366 and R491 were identified as key determinants of L-Glu binding. Mutation of R366 to Ala increases the K(d) for L-Glu by 160-fold and eliminates the positive cooperativity observed for the binding of L-Glu and ATP to the wild-type enzyme, based on a rapid equilibrium random mechanism of substrate binding. Unlike the wild-type enzyme, the R366A mutant enzyme was able to form product using the substrate analogue gamma-aminobutyric acid, suggesting that R366 interacts with the alpha-carboxylate of L-Glu. Mutation of R491 to Ala decreased k(cat) for ATP hydrolysis by 70-fold; however, dipeptide product was only formed in 5% of these turnovers. These data suggest that R491 stabilizes the phosphorylated gamma-carboxylate of L-Glu during nucleophilic attack by the L-Cys to form the dipeptide product. T323, R474, and R487 were predicted to be ATP binding determinants. Mutation of each of these residues to Ala increased the apparent K(m) for ATP by 20-100-fold while having only modest effects on k(cat) or the apparent K(m)'s for the other substrates. Finally, mutation of R179, a conserved residue that is present in gamma-GCS, but not in GS, increased the apparent K(m) for both L-Cys and L-Glu.
γ-谷氨酰半胱氨酸合成酶(γ-GCS)催化L-谷氨酸(L-Glu)和L-半胱氨酸(L-Cys)的ATP依赖性连接反应,这是三肽谷胱甘肽从头生物合成的第一步。最近有研究表明,γ-GCS是谷氨酰胺合成酶(GS)的结构同源物,这为构建γ-GCS活性位点模型提供了基础[Abbott等人(2001年)《生物化学杂志》276卷,42099 - 42107页]。以该模型作为定点诱变的指导,已在寄生原生动物布氏锥虫的酶中鉴定并表征了γ-GCS活性位点中的底物结合决定因素。R366和R491被确定为L-Glu结合的关键决定因素。基于底物结合的快速平衡随机机制,将R366突变为丙氨酸(Ala)会使L-Glu的解离常数(K(d))增加160倍,并消除L-Glu和ATP与野生型酶结合时观察到的正协同性。与野生型酶不同,R366A突变酶能够使用底物类似物γ-氨基丁酸形成产物,这表明R366与L-Glu的α-羧基相互作用。将R491突变为丙氨酸会使ATP水解的催化常数(k(cat))降低70倍;然而,在这些周转反应中只有5%形成了二肽产物。这些数据表明,在L-Cys进行亲核攻击形成二肽产物的过程中,R491稳定了L-Glu的磷酸化γ-羧基。T323、R474和R487被预测为ATP结合决定因素。将这些残基中的每一个突变为丙氨酸都会使ATP的表观米氏常数(K(m))增加20 - 100倍,而对k(cat)或其他底物的表观K(m)影响较小。最后,γ-GCS中存在但GS中不存在的保守残基R179的突变增加了L-Cys和L-Glu的表观K(m)。