Abstract
Alkylamines are oxidized by a number of different types of enzymes, and the low oxidation potentials favor 1-electron transfer processes and aminium radicals. We studied the mechanism of N-dealkylation by hemoproteins using the prototypic substrate N,N-dimethylaniline (with isotopic substitution on the methyl groups), since there were considerable data available from kinetic deuterium isotope studies suggesting alternative mechanisms for different hemoproteins (Miwa, G.T., Walsh, J.S., Kedderis, G.L., and Hollenberg, P. F. (1983) J. Biol. Chem. 258, 14445-14449). Cytochrome P450 2B1 (P450), chloroperoxidase, and several biomimetic metalloporphyrin systems showed low kinetic hydrogen isotope effects (D(V/K)intra < or = 2.0, T(V/K) < or = 3.4), and hemoglobin, horseradish peroxidase, and prostaglandin H synthase yielded high isotope effects (D(V parallel K)intra > or = 3.5, T(V/K) > or = 8.1), in agreement with previous studies. Dinnocenzo and Banach (Dinnocenzo, J. P., and Banach, T. E. (1989) J. Am. Chem. Soc. 111, 8646-8653) have provided evidence that the pKa for an alpha-hydrogen of the N,N-dimethylaniline aminium radical is approximately 9 and also estimated that the pKa for the 4-hydrogen of a 1,4-dihydropyridine aminium radical is approximately 3.5. The oxidations of two model 1,4-dihydropyridines showed only low kinetic hydrogen isotope effects with all of the enzymes examined (D(V/K) < or = 2.3, T(V/K) < or = 2.8). Aminium radicals derived from aminopyrine and N,N-dimethylthioanisole accumulated only with those hemoprotein systems showing the high isotope effects with N,N-dimethylaniline. We conclude that specific base catalysis of alkyl hydrogen removal from aminium radicals by the (Fe.O)2+ complex is a feature of some hemoproteins, including P450s, and that the lack of such catalysis in other hemoproteins is the basis of their high kinetic hydrogen isotope effects.
Highlights
From the DeDartment of Biochemistrv and Center inMolecular Toxicology, Vanderbilt University School of Medicine, Nashville, T&nessee,37232-0146 "
P450s, and that the lack of such catalysis in other recombination) isrequired by the observation that theoxygen hemoproteins is the basis of their high kinetic hydro- inthe derived carbonyl compound is derivedfrom l 8 0 2 gen isotope effects
They calculated a pK, of -3.5 for the 1-benzyl-1,4- 1989). If deprotonation of such aminium radicals is really a dihydronicotinamide cationradical. Since the acidityof such rate-limiting step and the basis for kinetichydrogen isotope a vinylogous aminium radical is nearly lo6 greater than that effects, we might expect the ratio of aminium radical of the PhNMe;’ species, we considered the possibility that, if formationtodealkylatedproductformationto reflect the base catalysis of deprotonation is of significance in amine isotope effects
Summary
HCHO andHzOwere recovered in the aqueous phase after extraction Kinetic Hydrogen Isotope Effects for PhNMez N-Demethof substrate a t basic pH andanalyzed by liquidscintillation spectrometry. HPLC-HPLC analyses for (i) PhNMe, demethylation, (ii) nifeto estimate the intrinsic kinetic deuterium isotope effect Dk. The values estimated were 7.9 for horseradish peroxidase (with C2H600H)8, .3 for prostaglandin H synthase, and 10.9 for hemoglobin. These are in threange of 1.6-2.9 and further support the general view that thesebiomimetic model systems catalyze -demethylationreactions with low kinetic hydrogen isotope effects. The rate of aminopyrine aminium radical formawe had observedthat horseradishperoxidase appeared to tion was greater than thaotf HCHO formation insome cases, catalyze theoxidation of nifedipine with a low kinetic hydro- consistent with theview that aminium radical deprotonation gen isotope effect (D( V / K )= 1.6,T( V / K )= 1.8)(Guengerich, is rate limiting and thseource of the isotope effects seen with. Colored aminopyrine cation radicals have been detected following formation by peroxidases and P450/amine/hydroperoxide systems (Griffin et al, 1980), and colored N,N-dime-
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