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Dihydroxycoumarin Probes the Active Site of SULT1A1. (Biochemistry/Molecular Biology).

Dihydroxycoumarin Probes the Active Site of SULT1A1. Sundari Chodavarapu* and Joe D. Beckmann, Alma College, Biochemistry Department, Alma, MI 48801

Phenol sulfotransferase (SULT1A1) catalyses sulfuryl group transfer from 3'phosphoadenosine-5'-phosphosulafe (PAPS) to phenolic xenobiotics such as 7-hydroxycoumarin (7HC). Interestingly, binding of 7HC to SULT1A1 shifted the emission of this ligand from 450 nm to 400 nm (280 nm excitation). Analysis of this 400 nm emission was complicated by overlapping emissions from unbound 7HC and SULT1A1 tryprophanyl groups. To overcome this difficulty, we searched for new fluorescent probes with structural similarity to 7HC. Experiments using 6,7-dihydroxycoumarin (DHC) demonstrated it to be a SULT1A1 acceptor substrate. Fluorescence of DHC (450 nm, 280 nm excitation) was enhanced approximately 20 fold upon addition of PAP and SULT1A1. This enhanced fluorescence was obliterated by the addition of dodecyl sulfate, suggesting that it was due to SULT1A1:DHC:PAP ternary complex. Spectrofluorometric titrations of SULT1A1 with DHC and PAP indicated biphasic binding characteristics of this enzyme with approximate Kd values of 0.05 [micro]M and 0.35 pM. This behavior is consistent with equilibrium dialysis measurements of 7HC binding, and indicates subunit interactions that restrict ligand binding by this dimeric enzyme. (NSF MCB 9905421)

* Indicates the person who presented the paper
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Publication:Michigan Academician
Article Type:Brief Article
Geographic Code:1USA
Date:Mar 22, 2002
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