Enzymatic properties of vesicle-reconstituted human cytochrome P450SCC (CYP11A1) differences in functioning of the mitochondrial electron-transfer chain using human and bovine adrenodoxin and activation by cardiolipin.
The recently reported heterologous expression and purification of both human cytochrome P450SCC and adrenodoxin [Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Haedlam, M.J. & Tuckey, R.C. (1998) Arch. Biochem. Biophys. 353, 109-115] has enabled us to perform studies with the membrane-reconstituted human enzymes to better understand the side-chain cleavage reaction in humans. Human P450SCC was successfully reconstituted into dioleoylphosphatidylcholine vesicles with and without cardiolipin and its enzymatic properties characterized in the membrane-bound state. Enhancement of the P450SCC activity and significant activation by cardiolipin were observed when human adrenodoxin instead of bovine adrenodoxin was used as electron donor. In the absence of cardiolipin, Km for cholesterol was decreased twice in the case of human adrenodoxin indicating enhanced cholesterol binding. On the other hand, in the presence of cardiolipin in the membrane both Km and V for cholesterol were decreased with human adrenodoxin as electron donor. Kinetic analysis of the interaction between human P450SCC and its redox partners provided evidence for enhanced binding of the human electron donor to human P450SCC indicated by both an increased V and decreased Kd for human adrenodoxin compared with the values with bovine adrenodoxin. Because no similar effects were observed in Tween 20 micelles, these results suggest that the phospholipid membrane may play an important role in the interaction of human adrenodoxin with human P450SCC.
Kisselev
P
P
Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Minsk, Belarus, Germany.
Tuckey
R C
RC
Woods
S T
ST
Triantopoulos
T
T
Schwarz
D
D
D000325:Adrenodoxin / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002308:Cardiolipins / Q000494:pharmacology*; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000302:isolation & purification / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008823:Micelles; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000378:metabolism; D011136:Polysorbates / Q000494:pharmacology; D013045:Species Specificity
D002308:Cardiolipins; D008823:Micelles; D010743:Phospholipids; D011136:Polysorbates; D000325:Adrenodoxin; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme