Identifying the Structure‐Function Activity of Human Cytosolic Sulfotransferase (SULT) 1C4

Abstract

Human cytosolic sulfotransferases (hSULTs) are phase II conjugation enzymes that play a critical role in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. SULTs transfer a sulfuryl‐moiety from the obligate donor PAPS (3′‐phosphoadenosine 5′‐phosphosulfate) to a suitable hydroxyl or amine group of the substrate. While some of the SULTs have been studied extensively, others, such as the SULT 1C family, remain poorly characterized. The objective of this study is to determine the role of hSULT1C4 in drug metabolism by identifying novel substrates and defining the substrate binding, selectivity, and activity of the SULT1C4 enzyme using molecular modeling and kinetic analysis. SULT1C4 was bacterially expressed and purified for in vitro kinetic analysis. Steady‐state kinetic parameters for 1‐naphthol were analyzed using four concentrations of 1‐naphthol (0.5, 1, 2.5, and 7.5 uM) and four concentrations of PAPS (0.5, 1.5, 5, 15 uM). The Km of 1‐naphthol was approximately 1 uM, whereas the data for PAPS suggests the existence of two Kms (1 uM and 12 uM). To determine if these two Kms are due to SULT1C4's dimeric protein state, two different dimerization domain mutants, SULT1C4‐K272A and SULT1C4‐V277E, have been generated. Native page gel analysis has shown that these mutations prevent dimerization resulting in monomeric SULT1C4 proteins. Our data suggests that the dimeric state of SULT1C4 influences PAPS’ ability to bind to the enzyme.Funding sources: NIH ES022606 and GM038953