Human Cytosolic Sulfotransferases

描述（由申请人提供）：硫酸化是许多内源性甾醇、儿茶酚胺、胆汁酸和甲状腺激素以及外源性物质和治疗药物的主要结合反应。由PAPS（专性磺酸盐供体）的结合引起的SULT 2A 1的结构变化显著调节底物选择性。PAPS的结合导致环3的结构重排（约100）。30 AA），其形成PAPS和受体底物结合袋的外表面，导致底物结合袋的体积减少超过50%。平面类固醇如脱氢表雄酮（DHEA）可以在存在和不存在PAPS的情况下结合，而体积较大的底物如雷洛昔芬只有在PAPS不结合时才能结合。因此，我们推测在细胞和组织中与PAPS的相互作用可能是调节胞质SULT家族底物选择性的重要因素。 SULT 2A 1是唯一的亚型，其在有和没有PAP结合的情况下具有良好解析的结构，允许在酶的这两种结构构象中模拟底物结合。同样如在SULT家族中观察到的，SULT 2A 1是二聚体，并且在增加浓度的DHEA的硫酸化期间显示底物抑制。我们已经表明，单体SULT 2A 1不显示底物抑制，这表明亚基相互作用在SULT 2A 1动力学调节中的作用。因此，我们建议1）研究结构重塑对SULT 2A 1底物反应性和动力学的作用; 2）分析二聚化对SULT 2A 1动力学性质的作用; 3）表征PAPS相关结构重排在人SULT 1A 1和SULT 1 E1底物识别和动力学中的作用。 公共卫生相关性：磺基转移酶（SULT）2A 1是一种主要的肝酶，参与内源性甾醇、胆汁酸以及治疗药物和许多外源性物质的硫酸化。SULT 2A 1在PAPS结合后发生结构重排，调节底物选择性以及酶的动力学特性。我们建议调查的结构变化和相互作用，以及与底物的选择性和动力学在SULT 2A 1在体外和在完整的培养细胞中的机制功能。

DESCRIPTION (provided by applicant): Sulfation is a major conjugation reaction for many endogenous sterols, catecholamines, bile acids and thyroid hormones as well as xenobiotics and therapeutic drugs. Structural changes in SULT2A1 elicited by the binding of PAPS, the obligate sulfonate donor, significantly regulate substrate selectivity. The binding of PAPS results in a structural rearrangement of loop3 (approx. 30 AA) that forms the outer surface of the PAPS and acceptor substrate binding pockets resulting a decrease of over 50% in the volume of the substrate binding pocket. Planar steroids such as dehydroepiandrosterone (DHEA) can bind in both the presence and absence of PAPS, whereas bulkier substrates such as raloxifene, can bind only if PAPS in not bound. Therefore, we hypothesize that interactions with PAPS in cells and tissues may be an important factor regulating substrate selectivity in the cytosolic SULT family. SULT2A1 is the only isoform for which well-resolved structures are available with and without PAP bound allowing for modeling of substrate binding in both these structural conformations of the enzyme. Also as observed within the SULT family, SULT2A1 is a dimer and shows substrate inhibition during the sulfation of increasing concentrations of DHEA. We have shown that monomeric SULT2A1 does not show substrate inhibition suggesting a role for subunit interactions in the regulation of SULT2A1 kinetics. Therefore, we propose to 1) investigate the role of structural remodeling on the substrate reactivity and kinetics of SULT2A1; 2) analyze the role of dimerization on the kinetic properties of SULT2A1; 3) characterize the role of PAPS- associated structural rearrangement in substrate recognition and kinetics for human SULT 1A1 and SULT1E1. PUBLIC HEALTH RELEVANCE: Sulfotransferase (SULT) 2A1 is a major hepatic enzyme involved in the sulfation of endogenous sterols, bile acids as well as therapeutic drugs and many xenobiotics. SULT2A1 undergoes a structural rearrangement upon PAPS binding that regulates substrate selectivity as well as the kinetic properties of the enzyme. We propose to investigate the structural changes and interactions as well as the mechanistic features associated with substrate selectivity and kinetics in SULT2A1 both in vitro and in intact cultured cells.