SULT1B1 dimerization provides the enzyme with increased thermal integrity and a higher affinity for cofactor PAPS (1141.11)

Abstract

Cytosolic sulfotransferases (SULTs) catalyze the conjugation of a multitude of compounds ranging from drugs and xenobiotics to environmental toxins. Though highly conserved, the twelve human SULT isoforms exhibit an array of substrate specificities that are difficult to predict. We aim to predict substrate specificity in silico with high accuracy, as it would be a more time and cost‐effective method of ligand screening. We must first understand the mechanism by which SULTs coordinate cofactor (PAPS) binding, substrate binding, PAP release, and product release. In general, SULTs contain 3 highly conserved groups of amino acid residues that are essential for either catalysis, PAPS binding, or dimerization. Though it is highly conserved in mammals, and proximal to the critical PAPS binding domain, the dimerization domain (DD) has not been linked to any kinetic function. To investigate functional aspects of the DD in vitro, we first bacterially expressed and purified WT hSULT1B1 and a DD point mutant (K266L). The mutant’s monomerization status was verified via gel filtration. Both WT and monomeric K266L‐SULT1B1 fully retained sulfation activity after incubation at 37°C for 90 minutes. However, upon incubation at 42°C, monomeric SULT1B1 lost over 90% of its enzymatic activity after 20 minutes, while dimeric SULT1B1 retained nearly 75% of its activity, suggesting the monomer is less thermo‐stable. The location of the DD suggests that it may also play a role in PAPS binding. The Km of PAPS for the monomer was 5. 2 µM while WT hSULT1B1 exhibited a Km of 1.34 µM as shown by a 1‐naphthol sulfation assay, while 1‐naphthol Km’s were unaltered. In a similar fashion, changes in protein intrinsic fluorescence showed that the WT Kd for PAPS was 332 nM while the monomer was 478 nM. Our data suggest that the dimerization of SULT1B1, and likely other SULTs, contributes to the thermal stability of the enzyme, while contributing to a higher affinity for PAPS binding.Grant Funding Source: NIH grant R01 GM38953 to CNF