Ibarra C, Nieslanik B S, Atkins W M
Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, Washington 98195-7610, USA.
Biochemistry. 2001 Sep 4;40(35):10614-24. doi: 10.1021/bi010672h.
Most cytosolic glutathione S-transferases (GSTs) exploit a hydrogen bond between an active site Tyr and the bound glutathione (GSH) cofactor to lower the pK(a) of the GSH and generate the nucleophilic thiolate anion, GS(-). In human (hGSTA1-1) and rat (rGSTA1-1) homologues, the active site Tyr-9 has a low pK(a) of 8.1-8.3, for which the functional significance is unknown. Crystal structures of GSTA1-1 suggest that weakly polar interactions between the electropositive ring edge of Phe-10 and the pi-cloud of Tyr-9, in the apoenzyme, could stabilize the tyrosinate anion and also modulate the pK(a) of GSH. Upon binding a product GSH conjugate, Phe-10 moves away from Tyr-9, allowing the highly dynamic C-terminus to "close" over the active site. To explore the role of Phe-10 in modulating the Tyr-9 pK(a) and in ligand binding, rGSTA1-1 mutants F10Y, F10L, and F10A were characterized. The pK(a)s of Tyr-9 in the apoenzymes were 8.2 +/- 0.2, 8.7 +/- 0.2, and 9.3 +/- 0.1, respectively, for F10Y, F10L, and F10A, compared to 8.3 +/- 0.2 for the "wild type". The experimentally determined pK(a)s qualitatively paralleled the energies required to remove a proton predicted by ab initio calculations using model compounds constrained to the coordinates of rGSTA1-1. The pK(a) of GSH in the binary complex was significantly less affected by these substitutions. In contrast, F220I and F220Y C-terminal mutations caused the pK(a) of Tyr-9 to decrease modestly. For the binary complex with S-hexyl-GSH, which induces the "closed" conformation, Tyr-9 retains a low pK(a) and the Phe-10 substitutions have significant effects. Presumably, Phe-10 plays a critical structural role in stabilizing the closed conformation. The mutations F10L and F10A also slowed the rate of GSH conjugate binding by 10-20-fold, as measured by stopped-flow fluorescence. The effects of Phe-10 substitution were large for both steps of the biphasic binding reaction, suggesting the importance of aromatic interactions throughout the reaction coordinate. A unified view of the C-terminal dynamics of GSTA1-1 is discussed, which emphasizes the coupling between Tyr-9 ionization, active site solvation, and C-terminal dynamics.
大多数胞质谷胱甘肽S-转移酶（GSTs）利用活性位点酪氨酸与结合的谷胱甘肽（GSH）辅因子之间的氢键来降低GSH的pK(a)并生成亲核硫醇阴离子GS(-)。在人源（hGSTA1-1）和大鼠源（rGSTA1-1）同源物中，活性位点酪氨酸-9的pK(a)较低，为8.1 - 8.3，其功能意义尚不清楚。GSTA1-1晶体结构表明，在脱辅酶中，苯丙氨酸-10的正电环边缘与酪氨酸-9的π电子云之间的弱极性相互作用可以稳定酪氨酸阴离子，同时也调节GSH的pK(a)。在结合产物GSH共轭物后，苯丙氨酸-10从酪氨酸-9移开，使得高度动态的C末端能够“闭合”覆盖活性位点。为了探究苯丙氨酸-10在调节酪氨酸-9的pK(a)以及配体结合中的作用，对rGSTA1-1突变体F10Y、F10L和F10A进行了表征。F10Y、F10L和F10A脱辅酶中酪氨酸-9的pK(a)分别为8.2±0.2、8.7±0.2和9.3±0.1，而“野生型”为8.3±0.2。实验测定的pK(a)在定性上与使用受rGSTA1-1坐标约束的模型化合物进行从头计算预测的去除质子所需能量平行。这些取代对二元复合物中GSH的pK(a)影响较小。相比之下，F220I和F220Y C末端突变导致酪氨酸-9的pK(a)适度降低。对于诱导“闭合”构象的与S-己基-GSH的二元复合物，酪氨酸-9保留较低的pK(a)，苯丙氨酸-10的取代有显著影响。据推测，苯丙氨酸-10在稳定闭合构象中起关键的结构作用。通过停流荧光测量，F10L和F10A突变也使GSH共轭物结合速率减慢了10 - 20倍。苯丙氨酸-10取代对双相结合反应的两个步骤影响都很大，表明在整个反应坐标中芳香族相互作用的重要性。本文讨论了GSTA1-1 C末端动力学的统一观点，强调了酪氨酸-9离子化、活性位点溶剂化和C末端动力学之间的耦合。
