title: Spectrofluorimetric analysis of 7-hydroxycoumarin binding to bovine phenol sulfotransferase.
abstract: Phenol sulfotransferases (SULT1s, EC 2.8.2.1) catalyze sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. Previous work with the bovine SULT1A1 has utilized the highly fluorescent substrate 7-hydroxycoumarin (7-HC, umbelliferone) as an acceptor substrate [Biochem. Biophys. Res. Commun. 261 (1999) 815]. Here we report that adenosine-3',5'-bisphosphate (PAP)-dependent binding of 7-HC to bSULT1A1 can be observed due to the appearance of a 400-420-nm shoulder in the emission spectrum, using an excitation wavelength of 280 nm. This emission was observed by placing 7-HC in ethanol, which is consistent with bSULT1A1 phenol binding site hydrophobicity. Titrations with 7-HC indicate a K(d) for 7-HC of 0.58 microM and substoichiometric binding to the homodimeric enzyme. The bSULT1A1:PAP:7-HC complex could be disrupted with pentachlorophenol (PCP), titrations with which indicated 0.5 equivalents per enzyme subunit. Titrations of enzyme plus 7-HC with PAP also indicated 0.5 equivalents per enzyme subunit. These results suggest a model of homodimeric bSULT1A1 in which subunit interactions favor half-site reactivity in the formation of a dead end complex.
authors.affiliation: Department of Biochemistry, Alma College, 614 West Superior Street, MI 48801, USA. beckmann@alma.edu
authors.forename: Joe D
authors.initials: JD
authors.lastname: Beckmann
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Burkett
authors.forename: Michelle
authors.initials: M
authors.lastname: Sharpe
authors.forename: Lenore
authors.initials: L
authors.lastname: Giannunzio
authors.forename: Dan
authors.initials: D
authors.lastname: Johnston
authors.forename: Sarah
authors.initials: S
authors.lastname: Abbey
authors.forename: Aaron
authors.initials: A
authors.lastname: Wyman
authors.forename: Lokman
authors.initials: L
authors.lastname: Sung
mesh_terms: D000818:Animals; D015239:Arylsulfotransferase; D002417:Cattle; D051381:Rats; D013050:Spectrometry, Fluorescence; D015238:Sulfotransferases / Q000737:chemistry* / Q000378:metabolism; D014468:Umbelliferones / Q000737:chemistry* / Q000378:metabolism
chemical_list: D014468:Umbelliferones; C031477:7-hydroxycoumarin; D015238:Sulfotransferases; D015239:Arylsulfotransferase; C117544:SULT1A1 protein, human