title: Comparative studies on the accessibility and functional importance of tyrosine residues in cytochrome P-450 isozymes.
abstract: Cytochromes P-450 LM2 and P-450 LM4 from rabbit liver microsomes were chemically modified with tetranitromethane. Nitration of two tyrosine residues of both isozymes inhibits the benzphetamine N-demethylase activity of P-450 LM2 as well as the p-nitrophenetole O-deethylase activity of P-450 LM4 by about 80%. For identification of the modified tyrosine residues the inactivated enzymes were digested with trypsin, and the labeled peptides were separated by HPLC. Sequencing of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM2 showed that the tyrosine residues at positions 235 and 380 were nearly fully nitrated, while Tyr-348, Tyr-484 and Tyr-111 were only partially labeled (about 40-50%). In the presence of the heme-binding inhibitor metyrapone, Tyr-380 is partially protected against modification, and the extent of inactivation is diminished as well. These results suggest that Tyr-380 of cytochrome P-450 LM2 presents a catalytically essential amino acid residue at its active center. Sequence analyses of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM4 revealed that mainly Tyr-243 and Tyr-271 were labeled, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent (about 30-45%). Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are suggested to be functionally involved in the interaction with NADPH-cytochrome P-450-reductase.
authors.affiliation: Central Institute of Molecular Biology, Academy of Sciences of the GDR, Berlin-Buch.
authors.forename: G R
authors.initials: GR
authors.lastname: Jänig
authors.forename: R
authors.initials: R
authors.lastname: Kraft
authors.forename: H
authors.initials: H
authors.lastname: Rabe
authors.forename: A
authors.initials: A
authors.lastname: Makower
authors.forename: K
authors.initials: K
authors.lastname: Ruckpaul
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D008297:Male; D008797:Metyrapone / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D011817:Rabbits; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000502:physiology*
chemical_list: D007527:Isoenzymes; D009566:Nitrates; D010455:Peptides; D014443:Tyrosine; D003577:Cytochrome P-450 Enzyme System; D014357:Trypsin; D008797:Metyrapone