Inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion (diethyl p-nitrophenyl phosphorothionate).
The mechanism of the inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion has been investigated with special reference to the covalent binding to the enzyme of the atomic sulfur (S) released upon oxidation of the compound. In addition to the previously reported loss of cytochrome P-450 detectable as its carbon monoxide complex, a loss of heme detectable as pyridine-hemochromagen, and a loss of enzymatic activity toward benzphetamine and ethoxycoumarin are shown. The loss of heme is 80% of the loss of cytochrome P-450, which in turn is less than the loss of benzphetamine demethylase activity, the turnover number of the modified enzyme being two-thirds that of the native based on the amount of cytochrome P-450 detectable as its carbon monoxide complex. Treatment with performic acid or dithiothreitol removes 75% of the 35S covalently bound to the cytochrome P-450 during parathion metabolism. However, the dithiothreitol treatment does not restore cytochrome P-450 detectable as its carbon monoxide complex or enzymatic activity. Likewise, no protection against the loss of heme, cytochrome P-450, or enzymatic activity is afforded by inclusion of 1 mM dithiothreitol during the incubation with parathion, although the amount of irreversibly bound sulfur, i.e. sulfur not dissociable by dithiothreitol, is significantly reduced. In the absence of dithiothreitol, 4 nmol of 35S become bound to the cytochrome P-450 for each nanomole of P-450 no longer detectable as its carbon monoxide complex. Evidence is presented for the covalent binding of sulfur to at least three other amino acids than cysteine. It is suggested that structural changes resulting from the covalent binding of atomic sulfur to cysteine residues are responsible for at least some of the loss of enzymatic activity observed.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010278:Parathion / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D010278:Parathion; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D004229:Dithiothreitol