Dynamics and functional activity of cytochrome P450scc selectively labeled with fluorescein isothiocyanate.
Studies have been done to assess the effect of selective chemical modification of Lys338 on the functional activity and conformational mobility of cytochrome P450scc. It is found that fluorescently labelled cytochrome P450scc retains cholesterol side-chain cleavage activity and the ability for spectral response of type I during interaction with cholesterol and protein-protein interaction with the electron donor, adrenodoxin. Based on the quenching of the FITC fluorescence by iodide after incorporation of the labelled heme protein into an artificial phospholipid membrane, the orientation of Lys338 was determined. By measuring the efficiency of resonance fluorescence energy transfer in an FITC-heme pair during interaction of the labelled cytochrome P450scc with substrate and adrenodoxin as well as under insertion of the heme protein into membrane the changes of inter-molecular distance between Lys338 and heme were registered that indicate a functional importance of conformational mobility of cytochrome P450scc. It is shown that chemical modification of Lys338 does not directly affect the catalytic activity of enzyme but results in a significant decrease of its stability. Increasing th content of inactivated form of cytochrome P450scc is followed by an increase in the calculated distance between Lys338 and heme. However, under stabilizing conditions the decrease of the indicated distance is demonstrated. It is suggested that Lys338, being not directly involved in formation of the active sites of cytochrome P450scc, indirectly participates in the biological functions of this heme protein, providing the necessary conformational mobility to the protein molecule in the present fragment of the polypeptide chain. The disturbance of this mobility by inserting of the bulky fluorescent label into cytochrome P450scc polypeptide chain results in decrease of stability (lability of the structure) of the whole protein molecule.
Lepesheva
G I
GI
Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Minsk.
Usanov
S A
SA
D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology; D011487:Protein Conformation; D011510:Proteolipids; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D011510:Proteolipids; C020485:proteoliposomes; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine