Abstract
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.
Footnotes
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Send reprint requests to: Dr. S. Fournel-Gigleux, URA Centre Nationale de Recherche Scientifique 1288, Faculty of Medicine, 54500 Vandoeuvre-lès-Nancy, France. E-mail:sfg{at}pharmaco-med.u-nancy.fr
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↵1 Current affiliation: Unité de Recherche Associé au Centre National de Recherche Scientifique 1288, Faculty of Medicine, 54500 Vandoeuvre-lès-Nancy, France.
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↵2 Current affiliation: Department of Biochemistry, University of Edinburgh, Edinburgh, EH8 9XD, UK.
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This work was supported by the Région Lorraine, by the Institut Fédératif de Recherche “Protéines,” by the Ministère de l’Education Nationale, and by National Institutes of Health Grant DK49715. This work is in partial fulfillment of the doctoral thesis of C.S. S.F.G. was the recipient of an INSERM-MRC senior research fellowship.
- Abbreviations:
- UGT
- UDP-glucuronosyltransferase
- 4-MU
- 4-methylumbelliferone
- DMEM
- Dulbecco’s modified Eagle’s minimum
- SDS
- sodium dodecyl sulfate
- PCR
- polymerase chain reaction
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- Received July 18, 1996.
- Accepted November 25, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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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
