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Phenylalanine 90 and 93 Are Localized within the Phenol Binding Site of Human UDP-Glucuronosyltransferase 1A10 as Determined by Photoaffinity Labeling, Mass Spectrometry, and Site-Directed Mutagenesis

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Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, LIMBP, Paul Verlaine University, Metz, France, Center for Biomedical Proteomics, Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507, and Viikki DDTC, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
Cite this: Biochemistry 2006, 45, 7, 2322–2332
Publication Date (Web):January 27, 2006
Copyright © 2006 American Chemical Society

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    4-Azido-2-hydroxybenzoic acid (4-AzHBA), a novel photoactive benzoic acid derivative, has been synthesized and used as a photoprobe to identify the phenol binding site of UDP-glucuronosyltransferases (UGTs). Analysis of recombinant His-tag UGTs from the 1A family for their ability to glucuronidate p-nitrophenol (pNP) and 4-methylumbelliferone (4-MU) revealed that UGT1A10 shows high activity toward phenols and phenol derivatives. Purified UGT1A10 was photolabeled with 4-AzHBA, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry. A single modified peak corresponding to amino acid residues 89−98 (EFMVFHAQWK) of UGT1A10 was identified. The attachment site of the 4-AzHBA probe was localized to the quadruplet Phe90-Met91-Val92-Phe93 using ESI LC−MS/MS. Sequence alignment revealed that the Phe90 and Phe93 are conserved in UGT1A7−10. Site-directed mutagenesis of these two amino acids was then followed by kinetic analysis of the mutants with two phenolic substrates, pNP and 4-MU, containing one and two planar rings, respectively. Using the combination of photoaffinity labeling, enzymatic digestion, MALDI-TOF and LC-MS mass spectrometry, and site-directed mutagenesis, we have determined for the first time that Phe90 and Phe93 are directly involved in the catalytic activity of UGT1A10 toward 4-MU and pNP.

     This work was supported in part by the NIH Grants DK51971, DK49715 (A.R.-P.), and CA908028 (R.R.D.), Tobacco Settlement Funds (A.R.-P.), and the Academy of Finland, Project 207535 (M.F.).

     University of Arkansas for Medical Sciences.


     Paul Verlaine University.

     Eastern Virginia Medical School.

     University of Helsinki.


     Corresponding author. Telephone:  501-686-5414. Fax:  501-603-1146. Email:  [email protected].

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