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.

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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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
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Okita, Richard T
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University of Alabama Birmingham
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