Catalytic Characterization of Human Microsomal Glutathione S-Transferase 2: Identification of Rate-Limiting Steps
Catalytic Characterization of Human Microsomal Glutathione S-Transferase 2: Identification of Rate-Limiting Steps
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DOI：
10.1021/bi3014104
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发表时间：
2013-03-12
期刊：
影响因子：
2.9
通讯作者：
Rinaldo-Matthis, Agnes
中科院分区：
文献类型：
--
作者：
Ahmad, Shabbir;Niegowski, Damian;Rinaldo-Matthis, Agnes
微粒体谷胱甘肽S-转移酶2（MGST 2）是一种与白三烯C-4合酶（LTC 4S）同源的17 kDa三聚体整合膜蛋白。已经表明MGST 2在缺乏LTC 4 S的细胞中催化促炎介质白三烯C-4（LTC 4）的生物合成。MGST 2的详细生物化学研究对于了解其细胞功能和作为LTC 4产生酶的潜在作用至关重要。在这里，我们已经通过稳态和预稳态动力学实验表征了纯化的MGST 2的底物特异性和催化性质。与具有8.7 X 10（5）M-1 s（-1）的催化效率的LTC 4S相比，具有1.8 X 10（4）M-1 s（-1）的催化效率的MGST 2在生产LTC 4方面效率低得多。然而，这两种酶显示出相似的30-40 μ M的K-M（LTA 4）。虽然LTC 4S每个酶单体具有一个活化的谷胱甘肽（GSH）（形成硫醇盐），但MGST 2三聚体似乎仅显示三分之一的硫醇盐活化位点反应性，这部分解释了其较低的催化效率。此外，MGST 2对几种脂质氢过氧化物显示出类似于0.2 μ mol min（-1）mg（-1）的GSH依赖性过氧化物酶活性。MGST 2而不是LTC 4S在催化亲电底物1-氯-2，4-二硝基苯（CDNB）和脂质过氧化产物4-羟基-2-壬烯醛与GSH的缀合中是有效的。使用停流前稳态动力学，我们的特点是充分的催化反应的MGST 2与CDNB和GSH作为底物，表现出一个初始的快速平衡结合的GSH，然后硫醇形成。仅在低GSH浓度下观察到CDNB-GSH缀合步骤的爆发动力学（硫醇根阴离子形成在这些条件下成为速率限制）。产物释放迅速且不限制总体反应。因此，一般而言，化学缀合步骤在生理GSH浓度下对MGST 2是限速的。MGST 2和LTC 4S表现出不同的催化和机械特性，分别反映了对广泛和特定生理功能的适应。
Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C-4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C-4 (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 X 10(5) M-1 s(-1), MGST2, with a catalytic efficiency of 1.8 x 10(4) M-1 s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar K-M(LTA4) of 30-40 mu M. 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 similar to 0.2 mu mol 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.