Mechanisms and Functions of Human Sulfotransferases

描述(申请人提供)：第二阶段药物代谢酶(硫磺)催化的硫酸盐化在调节不同的激素以及药物和其他外源物质的解毒方面是重要的。硫酸盐化还会导致前致癌物的生物活化，从而产生毒性效应。本研究项目的长期目标是了解人体排泄物的生物学功能，并探讨它们在生理和病理条件下与人类健康的相关性。本建议的具体目的如下：1.研究人硫磺的催化、底物抑制和产物抑制/激活的机制。提出的旁路有序机制和相关的替代机制将通过动力学分析、同位素交换、硫化活性中心氨基酸残基鉴定和定点突变进行研究。这些信息将对生物转化途径的预测具有重要意义。2.研究硫酸盐类药物对人体硫磺催化活性的影响。临床上重要的药物硫酸盐对人硫磺的催化活性的抑制和激活作用将被研究。大肠杆菌表达和纯化的人硫磺、人肠道胞浆以及人Hep G2和Caco-2细胞将用于这些研究。临床药物对人体活动的影响可能会干扰激素调节和异物解毒的正常生物功能。3.明确人SULT1E1的氧化调控机制。利用酶分析、Western印迹、RT-PCR、氨基酸修饰、定点突变、动力学分析、晶体结构分析和计算机模拟等方法，研究人SULT1E1在Hep G2和Caco-2细胞中的氧化调节和纯化的人SULT1E1的氧化还原硫醇调节机制。了解某些人体唾液的氧化调节知识对于了解唾液在生理和病理条件下的功能能力是很重要的。这项提议研究的是人类的。这些研究将对了解毒素的生物学功能，包括激素调节、药物代谢、异物解毒和致癌原的生物活化具有重要意义。这些知识对于理解药物的副作用、药物间的相互作用、药物的开发以及在癌症预防和病因中所起的潜在作用将是非常重要的。

DESCRIPTION (provided by applicant): Phase II drug metabolizing enzymes sulfotransferases (SULTs) catalyzed sulfation is important in the regulation of different hormones and the detoxification of drugs and other xenobiotics. Sulfation also leads to bioactivation of procarcinogens leading to toxic effect. The long-term goal of this research project is to understand human SULT biological functions and to investigate their relevance to human health under physiological and pathological conditions. Specific aims in this proposal are as follows: 1. To investigate mechanisms of catalysis, substrate inhibition, and product inhibition/activation of human SULTs. The proposed bypass ordered mechanism and related alternative mechanisms will be investigated using kinetic analysis, isotope exchange, sulfated active site amino acid residue identification, and site-directed mutagenesis. The information will have important implications for the prediction of biotransformation pathways. 2. To investigate the effect of sulfated drugs on human SULT catalytic activities. The inhibition and activation effect of clinically important drug sulfates on catalytic activities of human SULTs will be investigated. E. coli expressed and purified human SULTs; human intestinal cytosols; and human Hep G2 and Caco-2 cells will be used for these investigations. The effect of clinical drugs on human SULT activities may interfere SULT normal biological functions in hormone regulation and xenobiotic detoxification. 3. To define oxidative regulation mechanisms of human SULT1E1. Oxidative regulation of human SULT1E1 in Hep G2 and Caco-2 cells and redox thiol regulation mechanisms of purified human SULT1E1 will be investigated using enzyme assay, Western blot, RT-PCR, amino acid modification, site-directed mutagenesis, kinetic analysis, crystal structure analysis, and computer modeling methods. Knowledge on oxidative regulation of certain human SULT is important in understanding the ability of SULT functioning under physiological and pathological conditions. This proposal studies human SULTs. These studies will be significant in understanding SULT biological functions including hormone regulation, drug metabolism, xenobiotic detoxification and procarcinogen bioactivation. The knowledge will be important in understanding drug side effect, drug-drug interaction, drug development, and the potential roles SULTs play in cancer prevention and causation.