Details

Autor(en) / Beteiligte

• Raner, Gregory M
• Chiang, Eric W
• Vaz, Alfin D. N
• Coon, Minor J

Titel

Mechanism-Based Inactivation of Cytochrome P450 2B4 by Aldehydes:  Relationship to Aldehyde Deformylation via a Peroxyhemiacetal Intermediate

Ist Teil von

• Biochemistry (Easton), 1997-04, Vol.36 (16), p.4895-4902

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

1997

Quelle

MEDLINE

Beschreibungen/Notizen

• The inactivation of cytochrome P450 2B4 by aldehydes in a reconstituted enzyme system requires molecular oxygen and NADPH and is not prevented by the addition of catalase, superoxide dismutase, epoxide hydrolase, glutathione, or ascorbic acid. A strong correlation between loss of enzymatic activity and bleaching of the heme chromophore was established, and the inactivation was shown to be irreversible upon dialysis. In general, saturated aldehydes are more inhibitory than those with α,β-unsaturation, as indicated by the k inact values, and primary aldehydes are more potent inactivators than the structurally related secondary and tertiary aldehydes. Consistent with recent studies on catalytic specificity of the T302A mutant of this cytochrome [Vaz, A. D. N., Pernecky, S. J., Raner, G. M., & Coon, M. J. (1996) Proc. Natl. Acad. Sci. U.S. A. 93, 4644−4648], the rate of aldehyde deformylation, as determined by formation of the alcohol with one less carbon atom, is greatly stimulated over that of the wild-type enzyme. Of particular interest, the rate of oxidation of aldehydes to carboxylic acids is decreased with the mutant, whereas the rate of inactivation via heme destruction is enhanced. Furthermore, comparative deuterium isotope effects and the relative rates of inactivation and product formation suggest that the mechanism of aldehyde inactivation of P450 2B4 involves the deformylation reaction and is unrelated to carboxylic acid formation. Finally, in the reaction of P450 2B4 with 3-phenylpropionaldehyde, the formation of a heme adduct with a molecular weight corresponding to that of native heme plus 104 mass units confirms the loss of the carbonyl group from the aldehyde prior to reaction with the chromophore. We conclude that inactivation of P450 by aldehydes occurs via homolytic cleavage of a peroxyhemiacetal intermediate to give an alkyl radical that reacts with the heme.