Self-catalyzed inactivation of hepatic cytochrome P-450 by ethynyl substrates.

The following acetylenic substrates have been shown to mediate NADPH-dependent loss of cytochrome P450 on incubation with hepatic microsomes from phenobarbital-pretreated rats: 1-ethynylcyclohexanol, 1ethynylcyclopentanol, 3-methyl-1-pentyn-3-01, norethisterone, (1-methoxycyclohexyl)acetylene, 3-(2,4-dichlorophenoxy)-1-propyne, 3-(4-nitrophenoxy)-l-propyne, 3-phenoxy-l-propyne, 4-phenyl-l-butyne, 3phenyl-1-propyne, cyclohexylacetylene, acetylene, 3pentyn-2-01, 4-methyl-2-octyn-4-01, 2-hexyne, p+nylacetylene, and N-( 1,1-dimethylpropynyl)-3,5-d1chlorobenzamide. A 10-fold higher nominal concentration of the last two agents was required to obtain the same degree of enzyme loss observed with the other agents at a 1 mM concentration. In vivo administration of acetylene gas and nine of the monosubstituted acetylenes led to accumulation of abnormal hepatic pigments. Similar pigments were not observed in rats treated with disubstituted acetylenes. The pigments obtained with acetylene gas, norethisterone, and six other substrates, after isolation, methylation, and purification, exhibited essentially identical electronic absorption spectra. Field desorption mass spectrometric analysis of these eight pigments has established that each one gives a molecular ion which corresponds to the sum of the molecular weights of the dimethyl ester of protoporphyrin IX plus the substrate plus (probably) an oxygen atom. These results are used to formulate a destructive mechanism in which the enzyme prosthetic heme covalently binds to the substrate during attempted metabolism of the triple bond.