Lack of Substrate Inhibition in a Monomeric Form of Human Cytosolic SULT2A1

Abstract

Cytosolic sulfotransferases (SULTs) are important Phase II conjugation enzymes that transfer a SO3 group from 3′‐phosphoadionsine‐5′ phosophosulfate (PAPS) to an acceptor molecule. Substrate inhibition is a common feature of the SULT family, though the mechanism is not well understood. For purification SULT2A1 was expressed as a maltose binding protein (MBP) ‐ fusion protein. The MBP‐SULT2A1 fusion protein eluted as a monomer during gel exclusion chromatography but formed a homodimer after cleavage of the MBP moiety. Kinetic analysis of the MBP‐2A1 monomer and pure SULT2A1 dimer found that both enzymes had similar Km (1.6 and 1.4 μM, respectively) and Vmax (8.6 and 8.4 pmol/min/mol of enzyme, respectively) values; however, MBP‐2A1 did not show substrate inhibition with increasing dehydroepiandrosterone (DHEA) concentrations. Kd values for DHEA binding with SULT2A1 and MBP‐SULT2A1 were 0.6 and 0.7 μM respectively and for PAPS, 0.3 μM for both. These results suggest that the dimerization at the conserved KTVE motif is involved in substrate inhibition.