ARGININE-52 AND HISTIDINE-54 LOCATED IN A CONSERVED AMINO-TERMINAL HYDROPHOBIC REGION (LX2-R52-G-H54-X3-V-L) ARE IMPORTANT AMINO-ACIDS FOR THE FUNCTIONAL AND STRUCTURAL INTEGRITY OF THE HUMAN LIVER UDP-GLUCURONOSYLTRANSFERASE UGT1-ASTERISK-6

The hepatic UDP-glucuronosyltransferase UGT16 is actively involved in the glucuronidation of short and planar phenols in humans. Based on t he 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 locat ed in a conserved hydrophobic region of the variable aminoterminal dom ain of UGT. Arg52 was replaced by alanine (mutant R52A), and His54 was replaced by alanine or glutamine (mutants H54A and H54Q), The immunol ogical and catalytic properties of UGT16 and mutants were examined af ter stable expression in V79 cell lines. Immunoblots and immunoprecipi tation studies revealed that the mutant and UGT16 proteins were expre ssed in the microsomal membranes in similar amounts. However, replacem ent of His54 by glutamine led to a complete loss of activity toward 4- methylumbelliferone, and the V-max value was decreased 4-5-fold in the mutants R52A and H54A compared with the wild-type enzyme. The dissoci ation constants that characterize the binding of 4-methylumbelliferone and UDP-glucuronic acid to UGT16 were not greatly affected by the mu tations. Interestingly, H54Q was not recognized by specific antibodies to the amino-terminal portion of UGT16, thereby indicating that this amino acid was critical to antibody recognition. In contrast, the mut ants R52A and H54A could not be differentiated from the wild-type prot ein by pH optimum or thermal denaturation. Furthermore, these mutants were still sensitive to irreversible inhibition by diethyl pyrocarbona te and 2,3-butanedione, with second-order inactivation constant values similar to those obtained for UGT1B, Altogether, the strict conserva tion of His54 and Arg52 and the mutational analysis of these residues suggest that these amino acids in the hydrophobic amino-terminal conse nsus sequence LX2-R52-G-H54-X3-V-L are important for the function and the structure required for optimal catalytic efficiency of UGT16.