Identification by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding.
Utilizing site-directed mutagenesis and an Escherichia coli expression system for bovine cholesterol side chain cleavage cytochrome P450, lysine residues at 377 and 381 are found to play crucial roles in binding bovine adrenodoxin, required for transfer of electrons to mitochondrial P450s. These lysine residues are conserved among mitochondrial P450s and have been implicated previously by chemical modification studies as being important for adrenodoxin binding. In the present study, site-directed mutagenesis producing either neutral or positive amino acids at 377 or 381 has no effect on the structure of side chain cleavage cytochrome P450 as determined spectrally or on the enzymatic conversion of cholesterol to pregnenolone. However, the estimated Ks of adrenodoxin binding is increased approximately 150-600-fold depending on the particular mutation. Therefore these conserved positively charged residues in mitochondrial P450s are the key sites for adrenodoxin binding which is electrostatic in nature.
Wada
A
A
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Waterman
M R
MR
D000325:Adrenodoxin / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000325:Adrenodoxin; D002786:Cholesterol Side-Chain Cleavage Enzyme; D008239:Lysine