Mechanism of Enzymatic Reduction of Steroid Double Bonds

Abstract

The rate-limiting step in the enzymatic reduction of double bonds in some steroids was studied with the aid of deuterated water and NADPH labeled in the A-and B-position with deuterium. The following reactions were studied : conversion of progesterone into 5n-pregnane-3,20-dione, catalyzed by rat liver microsomes ; conversion of progesterone into 5113-pregnane-3,20-dione, catalyzed by a partially purified A4-3-oxosteroid 5p-reductase from rat liver, and conversion of 7-dehydrocholesterol into cholesterol, catalyzed by rat liver microsomes fortified with a desalted 100000 x g supernatant fluid. It was concluded that the rate-limiting step in the reductions catalyzed by the A4-3-oxosteroid 5113-reductase and the A4-3-oxosteroid 5n-reductase is the addition of a hydride ion from NADPH. Evidence was obtained to indicate that in the reaction catalyzed by 7-dehydrocholesterol reductase the protonization step could be ratelimiting.Reduction of steroid double bonds, catalyzed by different mammalian enzymes, is known to involve addition of a hydrogen from NADPH to one carbon atom and addition of a hydrogen from the enzyme or from the medium to the other carbon atom. The hydrogen derived from NADPH has been assumed to add as a hydride ion and the hydrogen from the enzyme or the medium as a proton. This mechanism has been established for the following reactions : 501-and 5p-reduction of the Ad-double bound in various A4-3-oxosteroids [2-61, reduction of the A7-double bond in 7-dehydrocholesterol [7], of the A14-double bond in 4,4-dimethylcholesta-8,14-dien- 3p-01 [8] and of the A2*-double bond in lanosterol [S]. 5a-Reduction of 3-0x0-A4-steroids and reduction of the A24-double bond in lanosterol [4,9] involve a cis addition of hydrogens, whereas the other reactions involve a trans addition [7,9-111. With the exception of the 5p-saturation of A4-3-oxosteroids [3,6] the hydride ion comes from the B-side of NADPH [4,5,7,8]. It has not been fully established whether the addition of the hydrogen from NADPH or the addition of the proton is the rate-limiting step in these reactions. Both possibilities have been suggested [7,12]. Wilton et al. have shown that when enzymatic reduction of the A 7 double bond in 7-dehydrocholesterol is carried out in a medium containing tritiated water, there is only a small incorpora- Nomenclature. [4A-ZH]NADPH is equivalent to 4R-[4-2H]-NADPH and [4B-2H]NADPH to 4S-[4-*H]NADPH[ 11. 7-Dehydrocholesterol, 5,7-choIestadien-3@-01. tion of tritium into the 8p-position of the steroid [7].It was suggested that the low extent of incorporation of tritium was due to an isotope effect, indicating that addition of a proton to the 8113-position is ratelimiting in the reaction. However, the low extent of incorporation of tritium observed could be due to insufficient equilibration between the labelled medium and a proton source in the microsomal enzyme. In previous studies it was shown that the transfer of tritium from [4A-SH]-and [4B-sH] NADPH to the 5 position of different A4-3-oxosteroids catalyzed by rat liv...

Horse-Liver Alcohol Dehydrogenase and Pseudomonas testosteroni 3(17)beta-Hydroxysteroid Dehydrogenase Transfer Epimeric Hydrogens from NADH to 17beta-Hydroxy-5alpha-androstan-3-one. An Exception to One of the Alworth-Bentley Rules

“…Rule 4 was apparently already broken by the observation that rat liver microsomal 3a-hydroxysteroid dehydrogenase catalyzes the transfer of the 4-pro3 hydrogen of NADH [13], while the soluble enzyme from the same tissue catalyzes the transfer of the 4-pro-R hydrogen of NADH [14].…”

Microbial Transformation of Cholesterol into Coprostanol

“…It is noteworthy that in contrast to thereactions catalyzed by mammalian 3-0x0-A4-steroid 5p-reductases and mammalian 3-oxo-A4-steroid 5a-reductase(s) [Q, 13,18,19], only a small isotope effect occurred in the transfer of tritium from the labelled co-factor to the steroid. It is possible that there are different rate-limiting steps in the reactions catalyzed by the microbial reductase preparation and by the mammalian reductases.…”

Transfer of Deuterium from [1‐2H2]Ethanol to Krebs Cycle and Related Acids of Rat Liver in vivo

“…Several of the reductions are not equilibrium reactions, and isotope effects may thus be of importance in determining the extent of deuterium incorporation. Marked isotope effects have been observed in the transfer of hydrogen isotopes from NADPH in reductions of the 3,4-double bond of steroids [43], and in the reduction of 3-hydroxymethyl-3-glutaryl coenzyme A [44]. The absence of deuterium incorporation in certain reductions of the 3,4-double bond may also be due to a different cellular localization of alcohol dehydrogenase and the steroid reductase [8,45].…”