Relationship between cytochrome P450 catalytic cycling and stability: fast degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) in hepatoma cells is abolished by inactivation of its electron donor NADPH-cytochrome P450 reductase.
Ethanol-inducible cytochrome P450 2E1 (CYP2E1) involved in the metabolism of gluconeogenetic precursors and some cytotoxins is distinguished from other cytochrome P450 enzymes by its rapid turnover (in vivo half-life of 4-7 h), with ligands to the haem iron, both substrates and inhibitors, stabilizing the protein. CYP2E1 is also known to have a high oxidase activity in the absence of substrate, resulting in the production of reactive oxygen radicals. We suggested that the rapid intracellular turnover of the enzyme may be partly due to covalent modifications by such radicals or to other changes during catalytic cycling, in which case the inhibition of electron supply from NADPH-cytochrome P450 reductase would be expected to stabilize the protein. Fao hepatoma cells, where CYP2E1 showed a half-life of 4 h upon serum withdrawal, were treated for 1 h with 0.3 microM diphenylene iodonium (DPI), a suicide inhibitor of flavoenzymes, which resulted in approximately 90% inhibition of the microsomal NADPH-cytochrome P450 reductase and CYP2E1-dependent chlorzoxazone hydroxylase activities. Subsequent cycloheximide chase revealed that the CYP2E1 half-life increased to 26 h. Neither the degradation rates of total protein, CYP2B1 and NADPH-cytochrome P450 reductase nor the cellular ATP level were affected by DPI under the conditions employed. These results demonstrate for the first time that the short half-life of CYP2E1 in vivo may be largely due to the rapid destabilization of the enzyme during catalytic cycling rather than to the intrinsic instability of the protein molecule.
Zhukov
A
A
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden. andzhu@ki.se
Ingelman-Sundberg
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis / Q000378:metabolism*; D004583:Electrons; D004790:Enzyme Induction; D004795:Enzyme Stability; D000431:Ethanol / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D009861:Onium Compounds / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D014407:Tumor Cells, Cultured
D009861:Onium Compounds; D000431:Ethanol; C007517:diphenyleneiodonium; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D019392:Cytochrome P-450 CYP2E1; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase