Chemical modification of cytochrome P-450 LM2. Characterization of tyrosine as axial heme iron ligand trans to thiolate.
Phenobarbital-inducible isozyme cytochrome P-450 LM2 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes has been modified with N-acetylimidazole and tetranitromethane. Up to four tyrosine residues of cytochrome P-450 LM2 are accessible to O-acetylation and to nitration. N-Demethylase activity, spectral dissociation constants and substrate binding kinetics of differently acetylated enzyme indicate the existence of two groups of accessible tyrosines also differing in their reactivity towards N-acetylimidazole. The fast-reacting tyrosine residue representing the first group is involved in the binding of the type II substrate aniline and appears to be located near the heme as shown by the protecting effect of the inhibitor metyrapone against modification, but obviously is not necessary for N-demethylation. Acetylation of one further tyrosine residue, however, caused an almost complete inhibition of the enzyme, indicating its involvement in the catalytic mechanism at the active center. Nitration of two tyrosine residues inactivates to about 20%. Obviously the third and fourth tyrosine residue are without functional importance. The experiments evidencing two functionally linked tyrosines are in line with HPLC analyses of tryptic peptides of cytochrome P-450 LM2 nitrated in the presence of metyrapone which gave evidence for the location of two distinct tyrosine residues in the active center. Nitration of tyrosine residues results in the partial formation of a hyperporphyrin spectrum of cytochrome P-450 LM2. Its appearance is prevented in the presence of metyrapone and can be reversed by reduction of the nitrotyrosinate .
D000107:Acetylation; D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D014443:Tyrosine
D007093:Imidazoles; D008024:Ligands; D014443:Tyrosine; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital; C012160:N-acetylimidazole