PT  - JOURNAL ARTICLE
AU  - Grover P. Miller
AU  - Cheryl F. Lichti
AU  - Agnieszka K. Zielinska
AU  - Anna Mazur
AU  - Stacie M. Bratton
AU  - Anna Gallus-Zawada
AU  - Moshe Finel
AU  - Jeffery H. Moran
AU  - Anna Radominska-Pandya
TI  - Identification of Hydroxywarfarin Binding Site in Human UDP Glucuronosyltransferase 1A10: Phenylalanine<sup>90</sup> Is Crucial for the Glucuronidation of 6- and 7-Hydroxywarfarin but Not 8-Hydroxywarfarin
AID  - 10.1124/dmd.108.022863
DP  - 2008 Nov 01
TA  - Drug Metabolism and Disposition
PG  - 2211--2218
VI  - 36
IP  - 11
4099  - http://dmd.aspetjournals.org/content/36/11/2211.short
4100  - http://dmd.aspetjournals.org/content/36/11/2211.full
SO  - Drug Metab Dispos2008 Nov 01; 36
AB  - 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 Km 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 Vmax (3- and 10-fold, respectively) and minimum changes in Km. Liquid chromatographytandem 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. The American Society for Pharmacology and Experimental Therapeutics