Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin.
Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.
Adamovich
T B
TB
Institute of Bioorganic Chemistry, BSSR Academy of Sciences, Minsk, U.S.S.R.
Pikuleva
I A
IA
Chashchin
V L
VL
Usanov
S A
SA
D000325:Adrenodoxin / Q000502:physiology; D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism / Q000502:physiology*; D005288:Ferredoxins / Q000378:metabolism; D008239:Lysine / Q000032:analysis / Q000502:physiology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D013387:Succinic Anhydrides
D005288:Ferredoxins; D010446:Peptide Fragments; D013387:Succinic Anhydrides; D000325:Adrenodoxin; C031801:succinic anhydride; D003577:Cytochrome P-450 Enzyme System; D008239:Lysine