Determination of the Reaction Mechanism of Fulvestrant Sulfation with Human SULT1A1 and SULT1E1

Cytosolic sulfotransferases (SULT) are important Phase II conjugation enzymes. SULTs catalyze the transfer of a sulfonate group from 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) to an acceptor substrate. Fulvestrant (FUL) is an estrogen receptor antagonist that has been detected as a sulfate conjugate in vivo . FUL sulfation activity was detected with two SULT isoforms, SULT1A1 and SULT1E1. Two‐substrate kinetic plots with PAPS determined the Km's for FUL as 4.2 and 0.23 μM, with Vmax's of 7.8 and 62.5 pmol * min −1 * mg −1 , for SULT1A1 and SULT1E1, respectively. Molecular docking of FUL in the active site of the SULT isoforms with PAPS bound showed that while FUL was able to dock to the SULT1E1‐PAPS complex; FUL did not dock in the SULT1A1‐PAPS complex. Molecular models were made of the PAPS free enzymes, based on the crystal structure SULT2A1 without PAPS. FUL docked into the models with energies of −11.2 kJ for SULT1E1 and −11.7 kJ for SULT1A1. Kd determinations of FUL were performed with intrinsic fluorescence spectroscopy gave a Kd of 2.3 μM for SULT1A1 and 0.19 μM for SULT1E1. When the enzymes were pre‐saturated with PAP, there was no change in the Kd for SULT1E1, but no binding was observed for FUL to the PAP‐SULT1A1 complex. These data demonstrate that in SULT1A1, FUL sulfation is an apparent ordered reaction mechanism with FUL as the leading substrate, whereas in SULT1E1 FUL sulfation has a random Bi Bi reaction mechanism.