Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450.
Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.
Hall
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LR
Department of Medicinal Chemistry, University of Kansas, Lawrence 66045-2506.
Hanzlik
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RP
D000577:Amides / Q000378:metabolism*; D000818:Animals; D001549:Benzamides / Q000378:metabolism; D001665:Binding Sites; D002934:Cinnamates / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium; D066298:In Vitro Techniques; D007700:Kinetics; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D000577:Amides; D001549:Benzamides; D002934:Cinnamates; C065069:N,N-dimethylhydrocinnamide; C038529:N,N-dimethylbenzamide; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D010089:Oxidoreductases, N-Demethylating