Deuterium isotope effect in the interaction of N-nitrosodimethylamine, ethanol, and related compounds with cytochrome P-450IIE1

Abstract

Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity and produces an isotope effect on its metabolism. Our previous results showed that deuteration causes a 5-fold increase in the apparent Km, but not the Vmax, for the demethylation and denitrosation of NDMA in microsomes. In the present work, we studied the nature of this deuterium isotope effect with several compounds using acetone-induced microsomes as a source of cytochrome P-450IIE1. In the microsomal N-nitrosodiethylamine deethylase reaction, NDMA and [2H6]NDMA were competitive inhibitors and displayed apparent Ki values of 59 and 441 mM, respectively, showing an isotope effect of 0.13. Similarly, in the p-nitrophenol hydroxylase reaction, a deuterium isotope effect of 0.21 on the Ki was observed. With acetone as an inhibitor for p-nitrophenol hydroxylase, the isotope effect on the Ki was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase. When the oxidation of ethanol, [1,1-2H2]ethanol, [2,2,2-2H3]ethanol, and [2H6]ethanol was compared, an isotope effect of about 5 was found in the Vmax/Km due to the deuteration of the methylene group (carbon 1) but not due to the methyl group. However, the Vmax was not affected. A corresponding deuterium isotope effect was observed in the Ki when these compounds were used as competitive inhibitors for the NDMA demethylase reaction. The results demonstrate that deuteration of NDMA, ethanol, and related compounds results in an increase in the Km or Ki with little change in the Vmax of P-450IIE1-catalyzed reactions. The molecular basis of this isotope effect is discussed.