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

Grover P. Miller, Cheryl F. Lichti, Agnieszka K. Zielinska, Anna Mazur, Stacie M. Bratton, Anna Gallus-Zawada, Moshe Finel, Jeffery H. Moran, Anna Radominska-Pandya

Research output: Contribution to journalArticle

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Abstract

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 Vmax (3- and 10-fold, respectively) and minimum changes in Km. 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.

Original languageEnglish (US)
Pages (from-to)2211-2218
Number of pages8
JournalDrug Metabolism and Disposition
Volume36
Issue number11
DOIs
StatePublished - Nov 2008
Externally publishedYes

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Binding Sites
Amino Acid Motifs
Tandem Mass Spectrometry
Site-Directed Mutagenesis
Liquid Chromatography
Anticoagulants
Cytochrome P-450 Enzyme System
Amino Acids
7-hydroxywarfarin
6-hydroxywarfarin
8-hydroxywarfarin
bilirubin uridine-diphosphoglucuronosyl transferase 1A10
Therapeutics

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

Cite this

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. / Miller, Grover P.; Lichti, Cheryl F.; Zielinska, Agnieszka K.; Mazur, Anna; Bratton, Stacie M.; Gallus-Zawada, Anna; Finel, Moshe; Moran, Jeffery H.; Radominska-Pandya, Anna.

In: Drug Metabolism and Disposition, Vol. 36, No. 11, 11.2008, p. 2211-2218.

Research output: Contribution to journalArticle

Miller, Grover P. ; Lichti, Cheryl F. ; Zielinska, Agnieszka K. ; Mazur, Anna ; Bratton, Stacie M. ; Gallus-Zawada, Anna ; Finel, Moshe ; Moran, Jeffery H. ; Radominska-Pandya, Anna. / 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. In: Drug Metabolism and Disposition. 2008 ; Vol. 36, No. 11. pp. 2211-2218.
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abstract = "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 Vmax (3- and 10-fold, respectively) and minimum changes in Km. 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.",
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AU - Lichti, Cheryl F.

AU - Zielinska, Agnieszka K.

AU - Mazur, Anna

AU - Bratton, Stacie M.

AU - Gallus-Zawada, Anna

AU - Finel, Moshe

AU - Moran, Jeffery H.

AU - Radominska-Pandya, Anna

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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 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 Vmax (3- and 10-fold, respectively) and minimum changes in Km. 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.

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