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*6.
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 the Vmax 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.
Senay
C
C
Unité de Recherche Associé au Centre National de la Recherche Scientifique 597, Université Henri Poincaré Nancy 1, France.
Ouzzine
M
M
Battaglia
E
E
Pless
D
D
Cano
V
V
Burchell
B
B
Radominska
A
A
Magdalou
J
J
Fournel-Gigleux
S
S
D000818:Animals; D001120:Arginine / Q000502:physiology*; D002460:Cell Line; D016384:Consensus Sequence; D006224:Cricetinae; D003412:Cricetulus; D005965:Glucuronates / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism*; D006639:Histidine / Q000502:physiology*; D006801:Humans; D006923:Hymecromone / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed
D005965:Glucuronates; D006923:Hymecromone; D006639:Histidine; D001120:Arginine; D014453:Glucuronosyltransferase