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Esculetin as a probe and substrate of bovine phenol sulfotransferase (bSULT1A1)
Author(s) -
Beckmann Joe D.,
DeGood Kate,
Cassidy Patrick,
Bauer Adam
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1008.6
Subject(s) - chemistry , fluorescence , sulfation , ternary complex , substrate (aquarium) , sulfotransferase , enzyme kinetics , stereochemistry , kinetics , ligand (biochemistry) , enzyme , active site , biochemistry , receptor , physics , oceanography , quantum mechanics , geology
Phenols are often conjugated by sulfotransferases (SULTs), with 3′‐phosphoadenosine−5′‐phosphosulfate (PAPS) as the donor substrate. Fluorescence energy transfer revealed a ternary complex of homodimeric bSULT1A1, PAP and 7‐hydroxycoumarin; however, unbound ligand signal confounded spectral interpretation. Therefore, we evaluated other (di)hydroxycoumarins as probes and substrates for recombinant bSULT1A1. 6,7‐Dihydroxycoumarin (DHC, esculetin) forms a highly fluorescent (λmax = 450 nm) PAP‐dependent complex with the enzyme. Titrations of DHC into SULT:PAP indicated half‐sites saturation with K d = 0.22 μM. Inclusion of borate, to model the sulfuryl transfer transition, resulted in full‐sites binding with K d = 1.7 μM. Reaction progress curves of DHC sulfation with excess PAPS were biphasic, emission spectra indicated two products, and initial rates indicated K m = 0.08 μM and k cat = 2.9 min −1 . Rapid kinetics of DHC binding is fit by two first‐order rates: [DHC]‐dependent (200 sec −1 ), and a slower [DHC]‐independent phase (25 sec −1 ). This may result from initial binding of DHC followed by a protein conformation change. Esculetin is thus providing new information regarding SULT catalysis and protein dynamics.