Structural requirements in 20-oxo-steroids for interaction with the binding site of 20beta-hydroxysteroid dehydrogenase.

Abstract

Kinetic measurements were made to investigate the interaction of 20β-hydroxysteroid dehydrogenase and a series of steroids with C-17 and/or C-21 hydroxyl groups, and the role of the region around C-17 and C-21 on the interaction of the reacting 20-oxo group with the catalytic site of the enzyme was considered. Substitution of a hydroxyl group for hydrogen at C-21 of 17-deoxy-steroid derivatives caused a significant decrease in the apparent Vmax value (to about one-sixth to one-ninth), but a slight increase in the apparent Km value (about 1.1- to 1.8-fold). The same change at C-21 of 17-hydroxy-steroid derivatives caused little or no decrease in the apparent Vmax value, but an increase in the apparent Km (about 1.4- to 1.8-fold) occurred which was similar to that with 17-deoxy-steroid derivatives. In 21-deoxy-11-deoxy-steroid derivatives, a 17α-hydroxyl substituent had little effect on the apparent Km value (about 0.8- to 1.1-fold) and produced a slight decrease in the apparent Vmax value (to about three-quarters to two-fifths). Introduction of a 17α-hydroxyl group into 21-deoxy-11-oxo-steroid derivatives led to a significant decrease in the apparent Km value (to about one-seventh) and a moderate decrease in the apparent Vmax value (to about five-sixths to one-half). Introduction of a 17α-hydroxyl group into 21-hydroxy-steroid derivatives caused the apparent Vmax value to increase by about 1.8- to 11-fold, but caused little or no decrease in the apparent Km value. These results suggest that the hydroxyl group at the 21- or 17α-position directly restricted the conformation and the orientation of the 20-oxo group towards the catalytic site of the enzyme and influenced the hydrogen transfer stage in the catalytic process, and also that, of the substituents at the 21- and 17α-positions, the latter may preferentially affect the reaction efficiency of the 20-oxo group. The presence of an oxo group at C-11 had an indirect influence on the effects of the substituents at the 21- and 17α-positions. The optimum orientation of the 20-oxo group for the catalytic reaction may occur in 21-deoxy-17-deoxy-11-deoxy-steroid derivatives. In an ideal ternary complex, the 20-oxo group of the steroid may project towards the β-face of the steroid ring and the conformation between the 20-oxo group and α-hydrogen of C-17 is nearly staggered, while that between C-21 and the α-hydrogen of C-17, looking along the C-17 to C-20 axis, is in a skew form ; the 20-oxo group is orientated rather far from the methyl group at the β-position of C-13 and more towards the β-chain of C-16.