17-HYDROXYLASE AND ANDIEN-BETA SYNTHETASE ACTIVITIES IN IMMATURE PIG TESTIS MICROSOMAL FRACTION - KINETIC-STUDIES OF THE PREGNENOLONE BINDING-SITE AND POSSIBLE INTERMEDIATES OF THE REACTIONS

The microsomal fraction from the testes of immature pigs can convert p regnenolone to 17-hydroxypregnenolone and also to 5,16-androstadien-3b eta-ol (andien-beta). The available evidence supports the hypothesis t hat both these reactions are catalysed by one enzyme, cytochrome-P450( 17alpha). In the absence of cytochrome b5, 17-hydroxypregnenolone will be the major product but that if cytochrome b5 is present in sufficie nt quantity, andien-beta becomes a major product. The point of diverge nce between the conversion of pregnenolone to either 17-hydroxypregnen olone or andien-beta was investigated using enzyme kinetic analysis to determine whether 16alpha-hydroxypregnenolone, 20beta-hydroxypregneno lone or 16-dehydropregnenolone could be specific intermediates to one reaction or the other. Product inhibition by 17-hydroxypregnenolone an d andien-beta was competitive for both 17-hydroxylase and ''andien-bet a synthetase'' supporting the current view of a common active site for both reactions. 16alpha-Hydroxypregnenolone was a very poor competiti ve inhibitor of 17-hydroxylase and andien-beta synthetase with K(i(app )) values many fold greater than the K(m(app)) for pregnenolone or the K(i(app)) for reaction product, rendering it unlikely that 16alpha hy droxylation is a key intermediary step in either pathway. 20beta-Hydro xypregnenolone was a more potent inhibitor of andien-beta synthetase t han of 17-hydroxylase and for the latter enzyme activity, the K(i(app) ) was lower than that for 17-hydroxypregnenolone itself. However, for andien-beta synthetase, 20beta-hydroxypregnenolone may be an early int ermediate as the K(i(app)) was consistent with the affinity for the ac tive site being intermediate between the K(m(app)) for pregnenolone an d the K(i(app)) for andien-beta. 16-Dehydropregnenolone was equipotent at inhibiting 17-hydroxylase and andien-beta synthetase activities su ggesting that 16-dehydropregnenes may be involved in the stages immedi ately prior to C21 side-chain cleavage.