Inhibition of Bovine Phenol Sulfotransferase (bSULT1A1) by CoA Thioesters

Abstract

Previous work with the bovine phenol sulfotransferase (bSULT1A1, EC ) demonstrated inhibition by CoA that was competitive with respect to the sulfuryl donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Leach, M., Cameron, E., Fite, N., Stassinopoulos, J., Palmreuter, N., and Beckmann, J. D. (1999) Biochem. Biophys. Res. Commun. 261, 815-819). Here we report that long chain acyl-CoAs are more potent inhibitors of bSULT1A1 and also of human dopamine sulfotransferase (SULT1A3) when compared with unesterified CoA and short chain-length acyl-CoAs. A complex pattern of inhibition was revealed by systematic variation of palmitoyl-CoA, PAPS, and 7-hydroxycoumarin, the acceptor substrate. Convex plots of apparent K(m)/V(max) versus [palmitoyl-CoA] were adequately modeled using an ordered rapid equilibrium scheme with PAPS as the leading substrate and by accounting for the possible binding of two equivalents of inhibitor to the dimeric enzyme. Interestingly, the first K(i) of 2-3 microm was followed by a second K(i) of only 0.01-0.05 microm, suggesting that positive subunit cooperativity enhances binding of long chain acyl-CoAs to this sulfotransferase. Simultaneous interaction of palmitoyl-CoA with both the nucleotide and phenol binding sites is suggested by two experiments. First, the acyl-CoA displaced 7-hydroxycoumarin from the highly fluorescent bSULT1A1.PAP.7-HC complex in a cooperative manner. Second, palmitoyl-CoA prevented the quenching of bSULT1A1 fluorescence observed with pentachlorophenol. Finally, titrations of bSULT1A1-pentachlorophenol complex with palmitoyl-CoA caused the return of protein fluorescence, and the binding of palmitoyl-CoA was highly cooperative (Hill constant of 1.9). Overall, these results suggest a model of sulfotransferase inhibition in which the 3'-phosphoadenosine-5'-diphosphate moiety of CoA docks to the PAPS domain, and the acyl-pantetheine group docks to the hydrophobic phenol binding domain.

Evaluation of Phenol Sulfotransferase (SULT1A1) Ligand Binding Order

“…5). Another proposed structural model of SULT function is PAPS-induced opening of both subunits [17], but this is contrary to these and other observations. Finally, the protection of SULT1A1 by bound nucleotide suggests a biological role in the longevity of the protein, if intracellular proteases and other protein turnover mechanisms are affected by its structure.…”

“…1), an approach used with other SULTs [13,25,27,32,46]. Indeed, fluorimetric titrations of some SULTs have been interpreted as proving random ligand binding [17,32]. In our case, the fluorescence responses were co-dependent on occupation of both the nucleotide and phenol binding sites (Fig.…”

“…This brought into question whether other reports of SULT ligand binding and kinetics might have suffered such an artifact. For example, fluorimetric titrations of human SULT1A1 with hydroxytamoxifen and 1-hydroxypyrene, both strong UV chromophores, may have come to erroneous conclusions [17]. Therefore, additional methods were necessary for this investigation.…”

“…The binding stoichiometries of 7-HC, PAP, and PCP all indicated "half sites" behavior, and so the models of SULT negative (anti) cooperativity and subunit oscillation during catalysis were first conceived. Others have since verified this anti-cooperativity by additional equilibrium titrations [17]. It is difficult, however, to envision much inter-subunit communication through the very limited SULT dimerization domain [18].…”