Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants.
Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy-Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability.
Hassett
C
C
Department of Environmental Health, School of Public Health and Community Medicine, University of Washington, Seattle 98195.
Aicher
L
L
Sidhu
J S
JS
Omiecinski
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA; D004851:Epoxide Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011110:Polymorphism, Genetic; D012313:RNA / Q000096:biosynthesis
D012313:RNA; D004247:DNA; D004851:Epoxide Hydrolases