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
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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DOI：
10.1021/bi0519001
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发表时间：
2006-02-21
期刊：
影响因子：
2.9
通讯作者：
Radominska-Pandya, A
中科院分区：
文献类型：
--
作者：
Xiong, Y;Bernardi, D;Radominska-Pandya, A
4-Azido-2-hydroxybeiizoic 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-glucuronosyl-transferases (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 UGTIA7-10. 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.
