ACTIVE SITE STUDIES OF HUMAN SULFOTRANSFERASES

磺基转移酶(ST)催化的硫酸盐化是含羟基(芳基或烷基)药物、致癌物、外源化合物和内源性化合物代谢的重要途径。药物和外源物质的硫酸盐化主要与解毒作用有关，通过解毒作用，相对疏水的化合物被生物转化为更容易排泄的水溶性硫酸酯。然而，有许多重要的例外，其中化学反应的硫酸酯的形成是导致有毒或致癌生物激活的代谢途径中的关键步骤。解毒或生物活化在很大程度上取决于所形成的各个硫酸酯产物的亲电反应性。这项研究的长期目标是确定人STS活性中心的结构特征，并研究STS催化硫化反应的催化机理。酶的催化机制和底物专一性取决于位于活性部位的特定氨基酸残基所产生的微环境和蛋白质分子的三维结构。根据我们以前的研究和文献报道，我们假设共底物PAPS结合位点由三个高度保守的区域和其他元件组成，不同STS的底物特异性由几个可变区的氨基酸残基决定。具体研究内容如下：1.利用光亲和探针对人肝酚-磺化ST(P-PST-1)、脱氢表雄酮ST(DHEA-ST)和雌激素ST(EST)的底物结合部位和PAPs结合部位的氨基酸残基进行鉴定。2.利用氨基酸修饰试剂对STS催化活性部位的必需氨基酸残基进行鉴定。3.人STS必需氨基酸和/或功能结构域的突变。4.人STS的计算分子结构研究。计算分子结构研究，结合特定目标1-3的实验结果，将有助于我们理解人STS的催化机制和不同底物的特异性。关于人类ST活性部位结构、底物特异性和催化机制的信息对于预测生物转化途径和内源化合物、外源化合物和致癌物代谢的个体差异具有重要意义。对STS活性部位的表征也将有助于寻找基于机理的特异性抑制剂/激活剂，用于研究STS的生理功能和设计新药。

Sulfotransferase (ST) catalyzed sulfation is an important pathway in the metabolism of hydroxyl (aryl or alkyl) containing drugs, carcinogens, xenobiotics and endogenous compounds. Sulfation of drugs and xenobiotics is mostly associated with detoxification by which a relatively hydrophobic compound is biotransformed into a more water-soluble sulfuric ester that is readily excreted. However, there are numerous important exceptions wherein the formation of chemically reactive sulfuric esters is an essential step in metabolic pathways leading to toxic or carcinogenic bioactivation. Detoxification or bioactivation is highly dependent upon the electrophilic reactivity of the individual sulfuric ester products formed. The long-term objective of the proposed research is to define the structural characteristics of the active sites of human STs and to investigate the catalytic mechanism of the sulfation reaction catalyzed by STs. An enzyme's catalytic mechanism and substrate specificity are determined by the micro-environment produced by specific amino acid residues localized within the active site and by the three dimensional structure of the protein molecule. According to our previous studies and reports in the literature, we hypothesize that the co-substrate PAPS binding site is composed of three highly conserved regions and other elements, and the substrate specificity of different STs is dictated by amino acid residues in several variable regions. Specific investigations to be carried out in this proposal are as follows: 1. Use photoaffinity probes to identify amino acid residues in both the substrate- binding site and PAPS binding site of human liver phenol- sulfating ST (P-PST-1), dehydroepiandrosterone ST (DHEA-ST), and estrogen ST (EST). 2. Use amino acid modification reagents to identify the essential amino acid residues in the catalytic active site of STs. 3. Mutagenesis of essential amino acids and/or functional domains of human STs. 4.Computational molecular structure studies of human STs. The computational molecular structure studies, combining the experimental results from specific aims 1-3, will help us to understand the catalytic mechanism and differing substrate specificity of human STs. The information on human ST active site structure, substrate specificity, and catalytic mechanism should have important implications for the prediction of biotransformation pathways and inter-individual differences in metabolism of endogenous compounds, xenobiotics, and carcinogens. The characterization of the active sites of STs will also help to find mechanism-based specific inhibitors/activators for use in studying the physiological functions of STs and designing new drugs.