Department of Medical Biochemistry and Biophysics, Chemistry II, Karolinska Institutet , Stockholm, Sweden.
Biochemistry. 2013 Mar 12;52(10):1755-64. doi: 10.1021/bi3014104. Epub 2013 Feb 27.
Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C4 (LTC4) in cells devoid of LTC4S. A detailed biochemical study of MGST2 is critical for the understanding of its cellular function and potential role as an LTC4-producing enzyme. Here we have characterized the substrate specificity and catalytic properties of purified MGST2 by steady-state and pre-steady-state kinetic experiments. In comparison with LTC4S, which has a catalytic efficiency of 8.7 × 10(5) M(-1) s(-1), MGST2, with a catalytic efficiency of 1.8 × 10(4) M(-1) s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar KM(LTA4) of 30-40 μM. While LTC4S has one activated glutathione (GSH) (forming a thiolate) per enzyme monomer, the MGST2 trimer seems to display only third-of-the-sites reactivity for thiolate activation, which in part would explain its lower catalytic efficiency. Furthermore, MGST2 displays GSH-dependent peroxidase activity of ∼0.2 μmol min(-1) mg(-1) toward several lipid hydroperoxides. MGST2, but not LTC4S, is efficient in catalyzing conjugation of the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the lipid peroxidation product 4-hydroxy-2-nonenal with GSH. Using stopped-flow pre-steady-state kinetics, we have characterized the full catalytic reaction of MGST2 with CDNB and GSH as substrates, showing an initial rapid equilibrium binding of GSH followed by thiolate formation. Burst kinetics for the CDNB-GSH conjugation step was observed only at low GSH concentrations (thiolate anion formation becoming rate-limiting under these conditions). Product release is rapid and does not limit the overall reaction. Therefore, in general, the chemical conjugation step is rate-limiting for MGST2 at physiological GSH concentrations. MGST2 and LTC4S exhibit distinct catalytic and mechanistic properties, reflecting adaptation to broad and specific physiological functions, respectively.
微粒体谷胱甘肽 S-转移酶 2（MGST2）是一种 17 kDa 的三聚体整合膜蛋白，与白三烯 C4 合酶（LTC4S）同源。有人认为，MGST2 在缺乏 LTC4S 的细胞中催化促炎介质白三烯 C4（LTC4）的生物合成。对 MGST2 的详细生化研究对于理解其细胞功能和作为 LTC4 产生酶的潜在作用至关重要。在这里，我们通过稳态和预稳态动力学实验对纯化的 MGST2 的底物特异性和催化特性进行了表征。与催化效率为 8.7×10(5)M(-1)s(-1)的 LTC4S 相比，催化效率为 1.8×10(4)M(-1)s(-1)的 MGST2 在产生 LTC4 方面效率明显较低。然而，两种酶对 LTA4 的 KM 值相似，为 30-40 μM。虽然 LTC4S 每个酶单体具有一个活化的谷胱甘肽（GSH）（形成硫醇盐），但 MGST2 三聚体似乎仅显示三分之一的活性位点用于硫醇盐活化，这在一定程度上解释了其较低的催化效率。此外，MGST2 对几种脂质过氧化物显示出依赖 GSH 的过氧化物酶活性，约为 0.2 μmol min(-1)mg(-1)。MGST2 能够有效地催化亲电子底物 1-氯-2,4-二硝基苯（CDNB）和脂质过氧化产物 4-羟基-2-壬烯与 GSH 的缀合，而 LTC4S 则不能。使用停流预稳态动力学，我们对 MGST2 与 CDNB 和 GSH 作为底物的完全催化反应进行了表征，表明 GSH 的快速初始平衡结合，然后形成硫醇盐。仅在低 GSH 浓度下观察到 CDNB-GSH 缀合步骤的爆发动力学（在这些条件下，硫醇盐阴离子形成成为限速步骤）。产物释放迅速，不会限制整个反应。因此，一般来说，在生理 GSH 浓度下，MGST2 的化学缀合步骤是限速步骤。MGST2 和 LTC4S 表现出不同的催化和机制特性，分别反映了对广泛和特定生理功能的适应。
