DEUTERIUM-ISOTOPE EFFECT IN THE INTERACTION OF NORMAL-NITROSODIMETHYLAMINE, ETHANOL, AND RELATED-COMPOUNDS WITH CYTOCHROME P-450IIE1

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 K(m), but not the V(max), 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 [H-2(6)]NDMA were competitive inhibitors and displayed apparent K(i) 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 K(i) was observed. With acetone as an inhibitor for p-nitrophenol hydroxylase, the isotope effect on the K(i) 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-H-2(2)]ethanol, [2,2,2-H-2(3)]ethanol, and [H-2(6)]ethanol was compared, an isotope effect of about 5 was found in the V(max)/K(m) due to the deuteration of the methylene group (carbon 1) but not due to the methyl group. However, the V(max) was not affected. A corresponding deuterium isotope effect was observed in the K(i) 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 K(m) or K(i) with little change in the V(max) of P-450IIEI-catalyzed reactions. The molecular basis of this isotope effect is discussed.