Cooperative ligand binding by bovine phenol sulfotransferase.
Although several phenol sulfotransferases (PSTs) that metabolize hormones and xenobiotics have been purified and examined by steady state kinetic methods, little is known about ligand binding and subunit interactions in these enzymes. Inhibition of a purified recombinant homodimeric bovine PST by 2,6-dichloro-4-nitrophenol (DCNP) and pentachlorophenol (PCP) displayed very sharp titration curves that required modeling with Hill equations with slope factors of 2 and 3, respectively. This observation suggested positive cooperative ligand binding during catalytic turnover. The binding of PCP was also monitored by intrinsic protein fluorescence, which was quenched up to 36% upon saturation with the inhibitor. In the absence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), quenching curves were fit with the Hill equation and an interaction factor of 1. In contrast, inclusion of PAPS increased the association of PCP and resulted in positive cooperative binding with an interaction factor of 1.6-1.9. Whereas adenosine-3',5'-diphosphate (PAP) also facilitated cooperative binding of PCP, adenosine-5'-monophosphate (AMP) was not effective. This correlated to inhibition of PST by PAP, whereas AMP was not inhibitory up to 1 mM. Therefore, occupation of the PST nucleotide binding site(s) facilitates a subunit interaction that can promote subsequent binding of phenolic ligands.
Beckmann
J D
JD
Department of Biochemistry, Alma College, MI 48801, USA.
Henry
T
T
Ulphani
J
J
Lee
P
P
D000818:Animals; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001667:Binding, Competitive; D002417:Cattle; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D009711:Nucleotides / Q000494:pharmacology; D010416:Pentachlorophenol / Q000378:metabolism / Q000494:pharmacology; D010636:Phenols / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism
D004791:Enzyme Inhibitors; D008024:Ligands; D009711:Nucleotides; D010636:Phenols; D011994:Recombinant Proteins; D010416:Pentachlorophenol; D015239:Arylsulfotransferase