Kinetic isotope effects in cytochrome P-450-catalyzed oxidation reactions. Intermolecular and intramolecular deuterium isotope effects during the N-demethylation of N,N-dimethylphentermine.
Two N,N-dimethylphentermine (N,N-dimethyl-2-amino-2-methyl-3-phenylpropane) substrates differing in deuterium substitution have been used to determine the intermolecular and intramolecular isotope effects associated with the cytochrome P-450-dependent N-demethylation of this substrate. No intermolecular isotope effect was observed in Vmax or Vmax/Km when the reaction rates for this substrate were compared to those for the substrate in which both N-methyl groups contained deuterium. In contrast, identical isotope effects of 1.6 to 2.0 were observed in both Vmax and Vmax/Km when this reaction was studied with a substrate in which only one of the two N-methyl groups was substituted with deuterium. Furthermore, both the intermolecular and intramolecular isotope effects were independent of the cytochrome P-450/NADPH-cytochrome P-450 reductase mole ratio. From these data, it is concluded that: 1) the carbon-hydrogen bond cleavage step does not contribute significantly to Vmax; 2) the contribution of the carbon-hydrogen bond cleavage step to Vmax is not detectably increased through changes in the cytochrome P-450/NADPH-cytochrome P-450 reductase mole ratio; 3) the N-methyl groups are free to exchange at the enzyme active site. The basis for these conclusions is the proposal of a new kinetic model for interpretation of intramolecular isotope effects which shows that intramolecular isotope effects are not necessarily equal to intrinsic isotope effects and, in fact, may be much smaller.
D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D008956:Models, Chemical; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010645:Phentermine / Q000031:analogs & derivatives* / Q000378:metabolism; D051381:Rats
C025982:N,N-dimethylphentermine; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D010645:Phentermine; D009251:NADPH-Ferrihemoprotein Reductase