Interaction of a spin-labelled cholesterol derivative with the cytochrome P-450scc active site.
The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytochrome P-450scc. Upon incubation with the cytochrome P-450scc electron transfer system, CNO is transformed to pregnenolone (Km = 33 microM, Vmax = 0.32 min-1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 microM). It binds tightly to cytochrome P-450scc as evidenced by a reversed type I spectral absorbance change (Kd = 5.9 microM) which is paralleled by a greater hyperfine splitting of the room-temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd = 1.9 microM). This finding is in accord with a rotational correlation time of about 10(-7) s, which is close to the tumbling rate of the protein. At 110 K the CNO-bound cytochrome P-450scc displays the ESR g-values gx = 2.404/2.456, gy = 2.245 and gz = 1.916; these are different from those of cholesterol-liganded cytochrome P-450scc and may thus serve as a marker for cytochrome P-450scc. Our data indicate that the stereospecificity of the cytochrome P-450scc side-chain-cleaving activity is not dependent on the nature of the cholesterol side-chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6-1.0 nm from the heme moiety.
Lange
R
R
INSERM U 128, Montpellier, France.
Maurin
L
L
Larroque
C
C
Bienvenüe
A
A
D000302:Adrenal Cortex / Q000032:analysis / Q000201:enzymology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000494:pharmacology; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000032:analysis*; D004579:Electron Transport; D008401:Gas Chromatography-Mass Spectrometry; D008928:Mitochondria / Q000032:analysis / Q000201:enzymology; D011284:Pregnenolone / Q000302:isolation & purification; D013113:Spin Labels
D013113:Spin Labels; C053027:25-doxylcholesterol; D011284:Pregnenolone; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme