Spectrofluorimetric analysis of 7-hydroxycoumarin binding to bovine phenol sulfotransferase

被引：8

作者：

Beckmann, JD ^{[1
]}

Burkett, RJ ^{[1
]}

Sharpe, M ^{[1
]}

Giannunzio, L ^{[1
]}

Johnston, D ^{[1
]}

Abbey, S ^{[1
]}

Wyman, A ^{[1
]}

Sung, L ^{[1
]}

机构：

[1] Alma Coll, Dept Biochem, Alma, MI 48801 USA

来源：

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS | 2003年 / 1648卷 / 1-2期

基金：

美国国家科学基金会;

关键词：

sulfotransferase; phenol; 7-hydroxycoumarin; PAP; fluorescence; cooperativity;

D O I：

10.1016/S1570-9639(03)00116-X

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Phenol sulfotransferases (SULT1s, EC 2.8.2.1) catalyze sulfinyl 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 muM 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. (C) 2003 Elsevier Science B.V. All rights reserved.

引用

页码：134 / 139

页数：6

共 29 条

[1] A FLUOROMETRIC ASSAY FOR QUANTITATING PHENOL SULFOTRANSFERASE ACTIVITIES IN HOMOGENATES OF CELLS AND TISSUES [J].