Functional characterization of four allelic variants of human cytochrome P450 1A2.
Human cytochrome P450 1A2 catalyzes important reactions in xenobiotic metabolism, including the N-hydroxylation of carcinogenic aromatic amines. In 2001, Chevalier et al. reported four new P450 1A2 sequence variants in the human population. We have now expressed these variants in Escherichia coli and measured protein expression (optical spectroscopy of holoenzyme and immunoblotting) and bioactivation of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and MeIQ (2-amino-2,4-dimethylimidazo[4,5-f]quinoline) in the lacZ reversion mutagenicity test. Enzyme kinetic analyses were performed for N-hydroxylation of five heterocyclic amine substrates and for O-deethylation of phenacetin. The most drastic effect was that of the R431W substitution: no holoenzyme was detectable. This residue is located in the "meander" peptide region and earlier site-directed mutagenesis studies demonstrated that it is critical for maintenance of protein tertiary structure. The other three variants had subtly different catalytic activities compared to the wild-type enzyme.
Zhou
Huijia
H
Department of Chemistry and Biochemistry, Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Guelph, Ont. N1G 2W1, Guelph, Canada.
Josephy
P David
PD
Kim
Donghak
D
Guengerich
F Peter
FP
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D007763:Lac Operon; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009152:Mutagenicity Tests / Q000379:methods; D017433:Protein Structure, Secondary; D011804:Quinolines / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011804:Quinolines; D011994:Recombinant Proteins; C029216:2-amino-3-methylimidazo(4,5-f)quinoline; D019388:Cytochrome P-450 CYP1A2; C036989:2-amino-3,4-dimethylimidazo(4,5-f)quinoline