Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity

Journal Article · Mon Nov 16 23:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00397a023· OSTI ID:5445889

Ekstroem, G; Norsten, C; Cronholm, T; Ingelman-Sundberg, M

Deuterium isotope effects (/sup D/(V/K)) and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled of (1,1-/sup 2/H/sub 2/) ethanol at various concentrations, and a competitive method, where 1:1 mixtures of (1-/sup 13/C)- and (/sup 2/H/sub 6/) ethanol or (2,2,2-/sup 2/H/sub 3/)- and (1,1-/sup 2/H/sub 2/) ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The /sup D/(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats. Similar isotope effects were reached with reconstituted membranes containing the rabbit ethanol-inducible cytochrome P-450 (LMeb), whereas control rat microsomes and membranes containing rabbit phenobarbital-inducible P-450 LM/sub 2/ oxidized the alcohol with /sup D/(V/K) of about 2.8 and 1.8, respectively. Addition of Fe/sup III/EDTA either to microsomes from phenobarbital-treated rabbits or to membranes containing P-450 LMeb significantly lowered the isotope effect. Incubations of all cytochrome P-450 containing systems of the xanthine-xanthine oxidase systems with (1R)- and (1S)-(1-/sup 2/H) ethanol, revealed, taking the isotope effects into account, that 44-66% of the ethanol oxidized had lost the 1-pro-R hydrogen. The data indicate that cytochrome P-450 dependent ethanol oxidation is not stereospecific and that cleavage of the C/sub 1/-H bond appears to be a rate-determining step in the catalysis by the ethanol-inducible form of P-450. The contribution of hydroxyl radicals in ethanol oxidation by the various enzymic systems is discussed.

• Research Organization: Karolinska Institute, Stockholm, Sweden
• OSTI ID: 5445889
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:23; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALCOHOL DEHYDROGENASE
ALCOHOLS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 13
CARBON ISOTOPES
CELL CONSTITUENTS
CHEMICAL REACTIONS
CYTOCHROMES
DEUTERIUM COMPOUNDS
DIGESTIVE SYSTEM
ENZYMES
ETHANOL
EVEN-ODD NUCLEI
GLANDS
HEMIACETAL DEHYDROGENASES
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
LIVER
MAMMALS
MICROSOMES
NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
OXIDATION
OXIDOREDUCTASES
PIGMENTS
PROTEINS
RABBITS
RATS
REACTION KINETICS
RODENTS
STABLE ISOTOPES
VERTEBRATES