Cytochrome b<inf>5</inf> augments the 17,20-lyase activity of human P450c17 without direct electron transfer

Abstract:

In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17α-hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19 sex steroids. Cytochrome b5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450- oxidoreductase (OR), and/or human cytochrome b5 in Saccharomyces cerevisiae and analyzed the 17α-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17α-hydroxylase and trace 17,20-lyase activities toward both Δ4 and Δ5 steroids. Coexpression of human OR with P450c17 increases the V(max) of both the 17α-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b5 with P450c17 also increases the V(max) of the 17,20-lyase reactions but not of the 17α- hydroxylase reactions. Simultaneous expression of human b5 with P450c17 and OR, or addition of purified human b5 to microsomes from yeast coexpressing human P450c17 and OR, further increases the V(max) of the 17,20-lyase reaction without altering 17α-hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b5, apo-b5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action of b5 does not require electron transfer from b5 to P450c17. These data suggest that human b5 acts principally as an allosteric effector that interacts primarily with the P450c17-OR complex to stimulate 17,20-lyase activity.

Año de publicación:

1998

Keywords:

Fuente:

scopus