Arginine residues in the active site of human phenol sulfotransferase (SULT1A1)
Arginine residues in the active site of human phenol sulfotransferase (SULT1A1)
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DOI：
10.1074/jbc.m306045200
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发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
Chen, XR
中科院分区：
文献类型：
--
作者：
Chen, GP;Chen, XR
胞液硫转移酶(STS)催化含羟基化合物的硫酸盐化反应。人酚硫转移酶(SULT1A1)是催化简单酚硫酸盐化的主要酶。由于其广泛的底物特异性和缺乏内源底物，SULT1A1的生物学功能被认为是一种重要的解毒酶。本报告采用氨基酸修饰、计算机结构模拟和定点突变等方法对SULT1A1活性部位的Arg残基进行了研究。Arg特异性修饰剂2，3-丁二酮以时间和浓度依赖的方式有效地灭活了SULT1A1，表明Arg残基在SULT1A1的催化活性中起着重要作用。根据计算机模型，Arg(78)、Arg(130)和Arg(257)对SULT1A1的催化活性可能是重要的。定点突变结果表明，Arg(78)上的正电荷对SULT1A1不是关键的，因为R78A仍然是活性的。相比之下，这个位置的负电荷R78E完全失活了SULT1A1。Arg(78)非常接近硫基转移的位置。Arg(257)与腺苷3‘-磷酸5’-磷酸硫酸盐(PAPS)中的3‘-磷酸非常接近。定点突变表明Arg(257)对SULT1A1是关键的：R257A和R257E都是无效的。虽然Arg(130)也离PAPs的3‘-磷酸很近，但R130A和R130E仍然具有活性，这表明Arg(130)不是SULT1A1催化活性的关键残基。计算机模拟表明，Arg(257)上的正电荷和3‘-磷酸上的负电荷之间的离子相互作用是稳定PAPs特异性结合的主要作用力。
Cytosolic sulfotransferases (STs) catalyze the sulfation of hydroxyl containing compounds. Human phenol sulfotransferase (SULT1A1) is the major human ST that catalyzes the sulfation of simple phenols. Because of its broad substrate specificity and lack of endogenous substrates, the biological function of SULT1A1 is believed to be an important detoxification enzyme. In this report, amino acid modification, computer structure modeling, and site-directed mutagenesis were used for studies of Arg residues in the active site of SULT1A1. The Arg-specific modification reagent, 2,3-butanedione, inactivated SULT1A1 in an efficient, time- and concentration-dependent manner, suggesting Arg residues play an important role in the catalytic activity of SULT1A1. According to the computer model, Arg(78), Arg(130), and Arg(257) may be important for SULT1A1 catalytic activity. Site-directed mutagenesis results demonstrated that the positive charge on Arg(78) is not critical for SULT1A1 because R78A is still active. In contrast, a negative charge at this position, R78E, completely inactivated SULT1A1. Arg(78) is in close proximity to the site of sulfuryl group transfer. Arg(257) is located very close to the 3'-phosphate in adenosine 3'-phosphate 5'-phosphosulfate ( PAPS). Site-directed mutagenesis demonstrated that Arg(257) is critical for SULT1A1: both R257A and R257E are inactive. Although Arg(130) is also located very close to the 3'-phosphate of PAPS, R130A and R130E are still active, suggesting that Arg(130) is not a critical residue for the catalytic activity of SULT1A1. Computer modeling suggests that the ionic interaction between the positive charge on Arg(257), and the negative charge on 3'-phosphate is the primary force stabilizing the specific binding of PAPS.