TY - JOUR T1 - Arginine 52 and Histidine 54 Located in a Conserved Amino-Terminal Hydrophobic Region (LX2-<em>R52</em>-G-<em>H54</em>-X3-V-L) Are Important Amino Acids for the Functional and Structural Integrity of the Human Liver UDP-Glucuronosyltransferase UGT1*6 JF - Molecular Pharmacology JO - Mol Pharmacol SP - 406 LP - 413 VL - 51 IS - 3 AU - Claire Senay AU - Mohamed Ouzzine AU - Eric Battaglia AU - Dorothy Pless AU - Virginie Cano AU - Brian Burchell AU - Anna Radominska AU - Jacques Magdalou AU - Sylvie Fournel-Gigleux Y1 - 1997/03/01 UR - http://molpharm.aspetjournals.org/content/51/3/406.abstract N2 - The hepatic UDP-glucuronosyltransferase UGT1*6 is actively involved in the glucuronidation of short and planar phenols in humans. Based on the irreversible inhibition of the enzyme on chemical modification by 2,3-butanedione and diethyl pyrocarbonate, the roles of His54 and Arg52 were investigated by oligonucleotide site-directed mutagenesis. These amino acids belong to a consensus sequence LX2-R52-G-H54-X3-V-L located in a conserved hydrophobic region of the variable amino-terminal domain of UGT. Arg52 was replaced by alanine (mutant R52A), and His54 was replaced by alanine or glutamine (mutants H54A and H54Q). The immunological and catalytic properties of UGT1*6 and mutants were examined after stable expression in V79 cell lines. Immunoblots and immunoprecipitation studies revealed that the mutant and UGT1*6 proteins were expressed in the microsomal membranes in similar amounts. However, replacement of His54 by glutamine led to a complete loss of activity toward 4-methylumbelliferone, and theVmax value was decreased 4–5-fold in the mutants R52A and H54A compared with the wild-type enzyme. The dissociation constants that characterize the binding of 4-methylumbelliferone and UDP-glucuronic acid to UGT1*6 were not greatly affected by the mutations. Interestingly, H54Q was not recognized by specific antibodies to the amino-terminal portion of UGT1*6, thereby indicating that this amino acid was critical to antibody recognition. In contrast, the mutants R52A and H54A could not be differentiated from the wild-type protein by pH optimum or thermal denaturation. Furthermore, these mutants were still sensitive to irreversible inhibition by diethyl pyrocarbonate and 2,3-butanedione, with second-order inactivation constant values similar to those obtained for UGT1*6. Altogether, the strict conservation of His54 and Arg52 and the mutational analysis of these residues suggest that these amino acids in the hydrophobic amino-terminal consensus sequence LX2-R52-G-H54-X3-V-L are important for the function and the structure required for optimal catalytic efficiency of UGT1*6. The American Society for Pharmacology and Experimental Therapeutics ER -