Oxidative cleavage of esters and amides to carbonyl products by cytochrome P450.
A series of esters and several amides were shown to undergo oxidative cleavage with the formation of carbonyl products in the presence of purified isoforms of liver microsomal cytochrome P450 (P450) in a reconstituted enzyme system. The reaction also requires NADPH and NADPH-cytochrome P450 reductase and is stimulated by phosphatidylcholine. Kinetic constants were determined in experiments in which the predicted aldehyde product was identified and quantitated by gas chromatography. A relationship was seen with P450 2E1 between the structures of the esters and the Vmax values, with the rates decreasing in the series of methyl formate to methyl valerate, and similarly in the series of methyl, ethyl, propyl, butyl, and amyl acetates. Furthermore, a clear correlation exists between the Km values of the ethyl esters examined and the log of the octanol/water partition coefficients of these substrates. With P450 2E1, the Km decreases significantly between one and four carbon atoms in the chain length of the acyl component of the ester but is unaffected by a further increase in length. However, no correlation was found between the Km value and the chain length of the alcohol moiety of the esters. Similarly, with P450 2B4 a large decrease in Km occurs between one and five carbons in the acyl component of the ethyl esters but is unaffected by a further increase in chain length. The observed correlation is presumed to arise from hydrophobic interactions between the access channel to the active site of P450 and the acyl side chain of the esters. P450 1A2 is also active in ester cleavage, and the three cytochromes examined with esters are active in the conversion of N-alkyl amides to aldehydes, as are P450s 2C3, 1A1, and 3A6. Studies on 2-butyl acetate oxidation by P450 2B4 in the presence of 18O2 showed 88% 18O incorporation into the product, 2-butanone. This is consistent with a mechanism that involves hydroxylation at the alpha-carbon of the alcohol component of the ester to yield an unstable geminal hydroxy ester, as proposed earlier by F. P. Guengerich et al. (1988, J. Biol. Chem. 263, 8176-8183) for several dihydropyridine carboxylic esters. Our results further indicate that such an intermediate decomposes by a nonhydrolytic mechanism and also rule out the possibility of transient ester hydrolysis with subsequent oxidation of the alcohol formed. In addition, they establish that oxidative cleavage is a widespread reaction among P450 cytochromes and commonly used esters and amides.
Peng
H M
HM
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606, USA.
Raner
G M
GM
Vaz
A D
AD
Coon
M J
MJ
D000577:Amides / Q000378:metabolism*; D000818:Animals; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004952:Esters / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology; D011817:Rabbits; D013379:Substrate Specificity
D000577:Amides; D004952:Esters; D010713:Phosphatidylcholines; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D009251:NADPH-Ferrihemoprotein Reductase