Cholesterol Side-chain Cleavage Enzyme - Mechanism of Action

Mechanism of Action

P450scc catalyzes the conversion of cholesterol to pregnenolone in three monooxygenase reactions. These involve 2 hydroxylations of the cholesterol side-chain, which generate, first, 22R-hydroxycholesterol and then 20alpha,22R-dihydroxycholesterol. The final step cleaves the bond between carbons 20 and 22, resulting in the production of pregnenolone and isocaproic acid.

Each monooxygenase step requires 2 electrons (reducing equivalents). The initial source of the electrons is NADPH. The electrons are transferred from NADPH to P450scc via two electron transfer proteins: adrenodoxin reductase and adrenodoxin. All three proteins together constitute the cholesterol side-chain cleavage complex.

The involvement of three proteins in cholesterol side chain cleavage reaction raises the question of whether the three proteins function as a ternary complex as reductase:adrenodoxin:P450. Both spectroscopic studies of adrenodoxin binding to P450scc and kinetic studies in the presence of varying concentrations of adrenodoxin reductase demonstrated that the reductase competes with P450scc for binding to adrenodoxin. These results demonstrated that the formation of a functional ternary complex is not possible. From these studies it was concluded that apparently the binding sites of adrenodoxin to its reductase and to P450 are overlapping and consequently, adrenodoxin functions as a mobile electron shuttle between reductase and P450. These conclusions have been confirmed by structural analysis of adrenodoxin and P450 complex.

The process of electron transfer from NADPH to P450scc is not tightly coupled, i.e. during electron transfer from adrenodoxin reductase via adrenodoxin to P450scc, a certain portion of the electrons leak outside of the chain and react with O₂ generating superoxide radicals. Steroidogenic cells include a diverse array of antioxidant systems to cope with the radicals generated by the steroidogenic enzymes.