Identification of hydroxywarfarin binding site in human UDP glucuronosyltransferase 1A10: Phenylalanine⁹⁰ is crucial for the glucuronidation of 6- and 7-hydroxywarfarin but not 8-hydroxywarfarin

Recent studies show that the extrahepatic human UDP-glucuronosyltransferase (UGT)1A10 is capable of phase II glucuronidation of several major cytochrome P450 metabolites of warfarin (i.e., 6-, 7-, and 8-hydroxywarfarin). This study expands on this finding by testing the hypothesis that the UGT1A¹⁰ F⁹⁰-M⁹¹-V⁹²-F⁹³ amino acid motif is important for proper recognition and conjugation of hydroxywarfarin derivatives. Site-directed mutagenesis studies demonstrate that F⁹⁰ is critical for 6- and 7-hydroxywarfarin glucuronidation based on the complete loss of enzymatic activity toward these substrates. In contrast, V92A and F93A mutants lead to higher rates of substrate turnover, have minimum changes in K _m values, and demonstrate substrate inhibition kinetics. A completely different activity profile is observed in the presence of 8-hydroxywarfarin. No change in either activity or affinity is observed with F90A when compared with wild type, whereas F93A and V92A mutants show increases in V_max (3- and 10-fold, respectively) and minimum changes in K_m. Liquid chromatography-tandem mass spectrometry studies show that enzymatic products produced by mutants are identical to wild-type products produced in the presence of 6-, 7-, and 8-hydroxywarfarin. Because F⁹⁰ is not critical for the glucuronidation of 8-hydroxywarfarin, there is likely another, different amino acid responsible for binding this compound. In addition, an inhibitory binding site may be formed in the presence of 6- and 7-hydroxywarfarin. This new knowledge and continued characterization of the hydroxywarfarin binding site(s) for UGT1A10 will help elucidate the molecular mechanism of hydroxywarfarin glucuronidation and potentially result in more effective anticoagulant therapies.