Abstract
The reactivities of 20β-hydroxysteroid dehydrogenase [EC 1.1.1.53] from Streptomyces hydrogenans towards 64 kinds of steroids having the pregnan-20-one skeleton were investigated. The presence and nature of a substituent around the reacting 20-oxo group of the steroid played a decisive role in the interaction with the catalytic site of the enzyme. In general, steroids having bulky group (s) at C-21 and/or C-17 did not act as substrates, though steroids having a bulky group at C-3 or C-11 were utilized by the enzyme. The existence of a substituent at C-16 caused 20-oxo steroids to lose their reactivity almost completely, but the existence of a substituent at C-3, C-6, C-9, C-11, C-17 or C-21 did not. The configurational relationship between the plane of the steroid ring and the C-17 side chain might also be important in steroid recognition by the enzyme. Among the nonsubstrate steroids, 16β-methylpregn-4-ene-3, 20-dione was a competitive inhibitor with respect to both steroid substrate and coenzyme. It was inferred that the steroid ring of the inhibitor molecule competed with the substrate binding process, while the 16β-methyl substituent competed with the coenzyme action. When 16α-methylpregn-4-ene-3, 20-dione, pregn-4-ene-3, 20-dione and 17, 21-dihydroxypregn-4-ene-3, 11, 20-trione were used as substrates, the apparent Km values for NADH were 13.97, 3.91 and 4.07μM, respectively. The presence of a substituent at C-16 unfavorably affected the apparent Km for NADH, but substituents at C-21, C-17 or C-11 did not. These results suggest that the coenzyme may be located in the vicinity of C-16 of the steroid molecule in the ternary complex, steroid-coenzyme-enzyme.
