UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body. In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER. Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP-GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain. The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.

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Cysteine residues in proteins and enzymes often fulfill rather important roles, particularly in the context of cellular signaling, protein-protein interactions, substrate and metal binding, and catalysis. At the same time, some of the most active cysteine residues are also quite sensitive toward (oxidative) modification. S-Thiolation, S-nitrosation, and disulfide bond and sulfenic acid formation are processes which occur frequently inside the cell and regulate the function and activity of many proteins and enzymes. During oxidative stress, such modifications trigger, among others, antioxidant responses and cell death. The unique combination of nonredox function on the one hand and participation in redox signaling and control on the other has placed many cysteine proteins at the center of drug design and pesticide development. Research during the past decade has identified a range of chemically rather interesting, biologically very active substances that are able to modify cysteine residues in such proteins with huge efficiency, yet also considerable selectivity. These agents are often based on natural products and range from simple disulfides to complex polysulfanes, tetrahydrothienopyridines, α,β -unsaturated disulfides, thiuramdisulfides, and 1,2-dithiole-3-thiones. At the same time, inhibition of enzymes responsible for posttranslational cysteine modifications (and their removal) has become an important area of innovative drug research. Such investigations into the control of the cellular thiolstat by thiol-selective agents cross many disciplines and are often far from trivial.

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.