Tsoi Carrie, Widersten Mikael, Morgenstern Ralf, Swedmark Stellan
Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden.
Biochem J. 2004 Mar 1;378(Pt 2):687-92. doi: 10.1042/BJ20031470.
The SULT (sulphotransferase) family plays a critical role in the detoxification and activation of endogenous and exogenous compounds as well as in the regulation of steroid hormone actions and neurotransmitter functions. The structure-activity relationships of the human SULTs have been investigated with focus on the amino acid 146 in hSULT1A3 and its impact on dopamine/PNP (p-nitrophenol) specificity. In the present study, we have generated canine SULT1D1 (cSULT1D1) variants with mutations at amino acid residues in the substrate-binding pocket [A146E (Ala-146-->Glu), A146D, A146Q, I86D or D247L]. These mutation sites were chosen with regard to their possible contribution to the marked dopamine/PNP preference of cSULT1D1. After characterization, we found that the overall sulphation efficiencies for the cSULT1D1 A146 and the I86 mutants were strongly decreased for both substrates compared with wild-type cSULT1D1 but the substrate preference was unchanged. In contrast, the D247L mutant was found to be more than 21-fold better at sulphating PNP (120-fold decrease in K(m) value) but 54-fold less efficient in sulphating dopamine (8-fold increase in K(m) value) and the preference was switched from dopamine to PNP, indicating the importance of this amino acid in the dopamine/PNP preference in cSULT1D1. Our results show that Asp-247 has a pronounced effect on the substrate specificity of cSULT1D1 and thus we have identified a previously unrecognized contributor to active-site selectivity.
磺基转移酶（SULT）家族在对内源性和外源性化合物的解毒与激活以及类固醇激素作用和神经递质功能的调节中发挥着关键作用。人们已经研究了人类SULT的构效关系，重点是hSULT1A3中的第146位氨基酸及其对多巴胺/p - 硝基苯酚（PNP）特异性的影响。在本研究中，我们构建了犬类SULT1D1（cSULT1D1）变体，其底物结合口袋中的氨基酸残基发生了突变[A146E（丙氨酸 - 146→谷氨酸）、A146D、A146Q、I86D或D247L]。选择这些突变位点是考虑到它们可能对cSULT1D1显著的多巴胺/PNP偏好性有贡献。经过表征，我们发现与野生型cSULT1D1相比，cSULT1D1的A146和I86突变体对两种底物的总体硫酸化效率均大幅降低，但底物偏好性未变。相反，发现D247L突变体在硫酸化PNP方面比野生型高出21倍以上（K(m)值降低120倍），但在硫酸化多巴胺方面效率低54倍（K(m)值增加8倍），并且偏好性从多巴胺转变为PNP，这表明该氨基酸在cSULT1D1的多巴胺/PNP偏好性中具有重要作用。我们的结果表明，天冬氨酸 - 247对cSULT1D1的底物特异性有显著影响，因此我们确定了一个以前未被认识到的活性位点选择性的影响因素。
