Mechanism of Enzymatic Reduction of Steroid Double Bonds

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 5α‐pregnane‐3, 20‐dione, catalyzed by rat liver microsomes; conversion of progesterone into 5β–pregnane‐3, 20‐dione, catalyzed by a partially purified Δ4‐3‐oxosteroid 5β‐reductase from rat liver, and conversion of 7‐dehydrocholesterol into cholesterol, catalyzed by rat liver microsomes fortified with a desalted 100000 ×gsupernatant fluid. It was concluded that the rate‐limiting step in the reductions catalyzed by the Δ4‐3‐oxosteroid 5β‐reductase and the Δ4‐3‐oxosteroid 5β‐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 rate‐limiting.