Influence of the structure of the A and B rings of various 20-oxo-steroids on their interaction with 20.BETA.-hydroxysteroid dehydrogenase.

Abstract

In order to investigate the functional role of the region around the A and B rings of 20-oxo-steroids in the interaction with the binding site of 20β-hydroxysteroid dehydrogenase, kinetic studies were made using 28 kinds of steroids which differed in the nature of the substituent, and in the shape and electronic character of the region around the A and B rings. Structural changes in the A ring of steroids, such as reduction of pregn-4-ene derivatives to the corresponding 5α- or 5β-pregnane derivatives, introduction of a further double bond into the A ring, and change of the 5α-series to 5β-series or change in the configuration and size of the substituent at C-3 caused only small changes in the apparent Km and Vmax values. It is suggested that the region around the A ring is of little significance in the interactions between a steroid molecule and the enzyme. It seems unlikely that the electronic character and shape of the B ring affect the steroid-enzyme interaction, since introduction of a C-5/C-6 double bond scarcely changed the kinetic constants. Neither a nonpolar (methyl group) nor a polar (hydroxyl group) substituent at the C-6 α-position (equatorial) produced any appreciable changes in the kinetic constants. Further, a 6-methyl group and a 9α-fluoro group (axial) did not cause any marked changes in the kinetic constants. On the other hand, introduction of a hydroxyl group at the C-6 β-position (axial) or removal of the 10β-methyl group caused a marked increase in the apparent Km. These findings suggest that recognition of the steroid moiety by the enzyme may involve the 10β-methyl group and the region around the β-side of the B ring. The nature of the interaction of these regions of the steroid with the enzyme may be hydrophobic, since a polar 6β-hydroxyl group had a marked repulsive effect on the steroid binding site of the enzyme, while a 10β-methyl group increased the binding affinity of the steroid.