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

Biochemistry. 2006 Feb 21;45(7):2322-32. doi: 10.1021/bi0519001.


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 Phe(90)-Met(91)-Val(92)-Phe(93) using ESI LC-MS/MS. Sequence alignment revealed that the Phe(90) and Phe(93) 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 Phe(90) and Phe(93) are directly involved in the catalytic activity of UGT1A10 toward 4-MU and pNP.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Azides / chemistry
  • Binding Sites
  • Chromatography, Liquid
  • Glucuronosyltransferase / antagonists & inhibitors
  • Glucuronosyltransferase / chemistry*
  • Glucuronosyltransferase / genetics*
  • Humans
  • Hymecromone / analogs & derivatives
  • Hymecromone / metabolism
  • Kinetics
  • Mass Spectrometry
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nitrophenols / metabolism
  • Phenol / metabolism
  • Phenylalanine / chemistry*
  • Photoaffinity Labels
  • Recombinant Proteins / metabolism
  • Salicylates / chemistry
  • Sequence Alignment
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • Azides
  • Nitrophenols
  • Photoaffinity Labels
  • Recombinant Proteins
  • Salicylates
  • Phenol
  • Hymecromone
  • Phenylalanine
  • 4-azidosalicylic acid
  • bilirubin uridine-diphosphoglucuronosyl transferase 1A10
  • Glucuronosyltransferase
  • 4-nitrophenol