Esculetin as a probe and substrate of bovine phenol sulfotransferase (bSULT1A1)

Abstract

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 Kd = 0.22 μM. Inclusion of borate, to model the sulfuryl transfer transition, resulted in full‐sites binding with Kd = 1.7 μM. Reaction progress curves of DHC sulfation with excess PAPS were biphasic, emission spectra indicated two products, and initial rates indicated Km = 0.08 μM and kcat = 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.