Enzymatic oxidation of ethyl carbamate to vinyl carbamate and its role as an intermediate in the formation of 1,N6-ethenoadenosine.
The carcinogen ethyl carbamate has been postulated to be activated by oxidation to vinyl carbamate and then to an epoxide which can react with nucleic acids [Dahl, G.A., Miller, J. A., and Miller, E. C. (1978) Cancer Res. 38, 3793-3804]. To date, the enzymatic conversion of ethyl carbamate to vinyl carbamate had not been demonstrated. Recently, we obtained evidence that the same cytochrome P-450 enzyme (P-450 2E1) is involved in the oxidation of both ethyl carbamate and vinyl carbamate [Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991) Chem. Res. Toxicol. 4, 168-179]. When human liver microsomes were incubated with NADPH and ethyl carbamate, the products vinyl carbamate, 2-hydroxyethyl carbamate, and ethyl N-hydroxycarbamate were detected by use of (a) combined capillary gas chromatography/chemical ionization mass spectrometry or (b) high-performance liquid chromatography of radioactive materials. A Km of approximately 54 microM was estimated for the conversion of vinyl carbamate to 1,N6-ethenoadenosine (in the presence of adenosine), but when the reaction was done with ethyl carbamate as the substrate, the rate of product formation was nearly first order in ethyl carbamate concentration (Km greater than 2 mM) and the rate was considerably slower than in the case of vinyl carbamate. The model derived with these parameters predicts a steady-state level of 0.22 microM vinyl carbamate, consonant with the value of approximately 0.2 microM estimated experimentally. A large kinetic deuterium isotope effect (greater than 7) was observed for the formation of 1,N6-ethenoadenosine from ethyl carbamate, and high isotope effects (6-8) were also noted for the formation of vinyl carbamate and 2-hydroxyethyl carbamate.(ABSTRACT TRUNCATED AT 250 WORDS)
Guengerich
F P
FP
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Kim
D H
DH
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D014520:Urethane / Q000031:analogs & derivatives* / Q000378:metabolism*
C007640:1,N(6)-ethenoadenosine; D014520:Urethane; C017963:vinyl carbamate; D003903:Deuterium; D000241:Adenosine