Roles of residues 129 and 209 in the alteration by cytochrome b5 of hydroxylase activities in mouse 2A P450S.
Cytochrome b5 stimulates the coumarin 7-hydroxylation activity of P450coh. A mutation of Arg-129 in P450coh, however, abolishes the stimulation. Moreover, this mutant P450coh binds loosely to cytochrome b5-conjugated Sepharose 4B, whereas wild-type P450coh binds tightly. Consistent with this, the mutation increases the Ka value for b5 binding approximately 6-fold. The identity of residue 209 also alters the stimulation of the activity of P450coh depending on the type of the substrates used and products formed. Coumarin 7-hydroxylation activity is greatly stimulated by cytochrome b5 only when Phe is at position 209, while cytochrome b5 stimulates testosterone hydroxylation activity of P450coh in which Phe, Asn, Ser or Lys substitutes residue 209. P450coh changes its rate of hydrogen peroxide formation depending on the identity of residue 209 and substrate used. Cytochrome b5 decreases the hydrogen peroxide formation of some P450coh whose activities are stimulated by the cytochrome; however, the decrease does not always result in stimulating the activity. The results indicate, therefore, that residues 129 and 209 play different roles in stimulating P450coh activity by cytochrome b5; Arg-129 is a key residue in the cytochrome b5-binding domain and is essential for the stimulation. Residue 209, however, alters the efficiency of electron transport for substrate oxidation as a residue which resides near the sixth ligand of heme and in the substrate-binding site.
Juvonen
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Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Iwasaki
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Negishi
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D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002846:Chromatography, Affinity; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry*; D004247:DNA; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data
D004247:DNA; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6