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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• 作者：Yan Xiong ; Dan Bernardi ; Stacie Bratton ; Michael D. Ward ; Eric Battaglia ; Moshe Finel ; Richard R. Drake ; and Anna Radominska-Pandya
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：February 21, 2006
• 年：2006
• 卷：45
• 期：7
• 页码：2322 - 2332
• 全文大小：257K
• 年卷期：v.45,no.7(February 21, 2006)
• ISSN：1520-4995

文摘

4-Azido-2-hydroxybenzoic acid (4-AzHBA), a novel photoactive benzoic acid derivative, hasbeen 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 toglucuronidate p-nitrophenol (pNP) and 4-methylumbelliferone (4-MU) revealed that UGT1A10 showshigh 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 localizedto the quadruplet Phe⁹⁰-Met⁹¹-Val⁹²-Phe⁹³ using ESI LC-MS/MS. Sequence alignment revealed that thePhe⁹⁰ and Phe⁹³ are conserved in UGT1A7-10. Site-directed mutagenesis of these two amino acids wasthen followed by kinetic analysis of the mutants with two phenolic substrates, pNP and 4-MU, containingone 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 thefirst time that Phe⁹⁰ and Phe⁹³ are directly involved in the catalytic activity of UGT1A10 toward 4-MUand pNP.