Dietze E C, Wang R W, Lu A Y, Atkins W M
University of Washington, Medicinal Chemistry, Seattle, Washington 98195-7610, USA.
Biochemistry. 1996 May 28;35(21):6745-53. doi: 10.1021/bi9530346.
A conserved tyrosine plays a critical role in catalysis by mammalian glutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, by forming a hydrogen bond to and stabilizing the thiolate form of glutathione. The hydrogen bonding properties of this tyrosine in the rat A1-1 GST (Tyr-9), in the absence and presence of ligands, have been studied by steady state and time-resolved fluorescence spectroscopy. In order to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalanine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these mutants have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation--emission spectra of the W21F mutant and the W21F-Y9F indicates that a red-shifted emission component is contributed by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-free enzyme. The pH-dependence of the intensity of these spectral cross-peaks is consistent with an active site tyrosine with a pKa of 8.1-8.3. Upon addition of GSH, the red-shifted component is quenched. Multifrequency phase/modulation fluorescence experiments qualitatively demonstrate that GSH causes a decrease in the average excited state lifetime on the red-edge of the spectrum of W21F but not of the W21F:Y9F spectrum. Steady state correlated difference spectra (W21F-W21F:Y9F) have been used to obtain a model for the excitation-emission correlated spectrum of Tyr-9, which indicates that Tyr-9 is heterogeneous at pH 7.5, with properties of both tyrosinate and "normal tyrosine". The tyrosinate fraction is eliminated, and the blue-shifted component becomes more intense upon addition of GSH conjugates, indicating that the weak hydrogen bond between Tyr-9 and thioethers has little charge-transfer character. The S-methyl GSH yields an "anomalous" spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. These results indicate that, in the absence of ligand, Tyr-9 forms a strongly polarized hydrogen bond or a fraction of the phenolic hydroxyl group is partially deprotonated. However, when a GSH conjugate with a sufficiently large hydrophobic group occupies the H-site, Tyr-9 is fully protonated, with little charge-transfer character.
一个保守的酪氨酸在α-、μ-和π-类哺乳动物谷胱甘肽S-转移酶（GSTs）的催化过程中起着关键作用，它通过与谷胱甘肽的硫醇盐形式形成氢键并使其稳定。通过稳态和时间分辨荧光光谱研究了大鼠A1-1 GST（Tyr-9）中该酪氨酸在无配体和有配体情况下的氢键性质。为了实现这一点，将大鼠A1-1 GST中发现的单个色氨酸（Trp 21）替换为荧光沉默的苯丙氨酸（W21F）。此外，构建了一个缺乏该色氨酸和催化酪氨酸的双突变体（W21F:Y9F），这些突变体已被用作Tyr-9处配体效应的探针。W21F突变体和W21F-Y9F的相关激发-发射光谱比较表明，在无配体的酶中，Tyr-9贡献了一个红移发射成分，其激发带在255和300 nm处。这些光谱交叉峰强度的pH依赖性与pKa为8.1-8.3的活性位点酪氨酸一致。加入谷胱甘肽后，红移成分被淬灭。多频相位/调制荧光实验定性地证明，谷胱甘肽导致W21F光谱红边的平均激发态寿命缩短，但不影响W21F:Y9F光谱。稳态相关差谱（W21F-W21F:Y9F）已被用于获得Tyr-9的激发-发射相关光谱模型，这表明Tyr-9在pH 7.5时是异质的，具有酪氨酸盐和“正常酪氨酸”的性质。加入谷胱甘肽共轭物后，酪氨酸盐部分被消除，蓝移成分变得更强，这表明Tyr-9与硫醚之间的弱氢键几乎没有电荷转移特征。S-甲基谷胱甘肽在pH 7.5时产生“异常”光谱，该光谱保留了与离子化酪氨酸盐一致的交叉峰。这些结果表明，在无配体的情况下，Tyr-9形成了一个强极化的氢键，或者酚羟基的一部分被部分去质子化。然而，当一个带有足够大疏水基团的谷胱甘肽共轭物占据H位点时，Tyr-9完全质子化，几乎没有电荷转移特征。
