Mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: characterization of reactive intermediate adducts to the heme and apoprotein.
The kinetics for the inactivation of cytochrome P450 2E1 and the mutant P450 2E1 T303A by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) were investigated. The two acetylenes inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified rabbit P450s 2E1 and 2E1 T303A in a reconstituted system in a time-, concentration-, and NADPH-dependent manner. The K(I) values for the inactivation of P450s 2E1 and 2E1 T303A by tBA were 1.0 and 2.0 mM, the k(inact) values were 0.20 and 0.38 min(-)(1), and the t(1/2) values were 3.5 and 1.8 min, respectively. The K(I) values for the tBMP-inactivated P450s were 0.1 and 1.0 mM, the k(inact) values were 0.12 and 0.07 min(-)(1), and the t(1/)(2) values were 5.9 and 10.2 min, respectively. Losses in enzyme activity occurred with concurrent losses in the P450 CO spectrum and P450 heme, which were accompanied by the appearance of two different tBA- or tBMP-modified heme products in each inactivated sample. LC-MS analysis of the adducts showed masses of 661 or 705 Da, consistent with the mass of an iron-depleted heme plus the masses of a tBA or tBMP reactive intermediate and one oxygen atom, respectively. Only the tBA-inactivated P450 2E1 revealed a tBA-adducted apoprotein with an increase in mass of 99 Da, corresponding to the mass of tBA plus one oxygen atom. Surprisingly, the inactivation, CO spectral and heme loss, and heme adduct formation of the tBA-inactivated T303A mutant were completely reversible after dialysis. In addition, metabolism of para-nitrophenol was not compromised by the tBA-inactivated T303A mutant. Therefore, our studies on the inactivation of P450s 2E1 and 2E1 T303A by tBA and tBMP suggest the existence of three distinct mechanisms for inactivation, among which includes a novel, reversible heme alkylation that has not been previously described with P450 enzymes.
Blobaum
Anna L
AL
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, USA.
Kent
Ute M
UM
Alworth
William L
WL
Hollenberg
Paul F
PF
D000114:Acetylene / Q000031:analogs & derivatives / Q000494:pharmacology*; D000478:Alkylation / Q000187:drug effects; D019943:Amino Acid Substitution; D000818:Animals; D001059:Apoproteins / Q000032:analysis / Q000737:chemistry*; D002248:Carbon Monoxide / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000235:genetics; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006418:Heme / Q000032:analysis / Q000737:chemistry*; D006900:Hydroxylation / Q000187:drug effects; D007700:Kinetics; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D001059:Apoproteins; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006418:Heme; D009249:NADP; D002248:Carbon Monoxide; D019392:Cytochrome P-450 CYP2E1; D000114:Acetylene