On the Rate‐Limiting Step in Microsomal Hydroxylation of Steroids

The rate‐limiting step in the microsomal, NADPH‐dependent 24‐ and 26‐hydroxylations of 5β‐cholestane‐3α,7α,12α‐triol, 6β‐hydroxylation of testosterone and 16α‐hydroxylation of pregnenolone has been studied. Possible isotope effects were determined when the hydrogen in the substrate which is removed in the hydroxylation was substituted with deuterium; when the 4A‐ or 4B‐hydrogen in NADPH was substituted with deuterium; and when the incubation was carried out in deuterated water.1 6β‐Hydroxylation of [6β‐ 2 H, 1,2‐ 3 H 2 ]‐plus [4‐ 14 C]labeled testosterone and 26‐hydroxylation of 5β‐[26‐ 2 H 3 ]‐cholestane‐3α,7α,12α‐triol did not involve significant isotope effect, whereas 16α‐hydroxylation of [16α‐ 2 H, 3α‐ 3 H]‐plus [4‐ 14 C]‐labeled pregnenolone and 24‐hydroxylation of 5β‐[24‐ 2 H 2 ]cholestane‐3α,7α,12α‐triol involved isotope effects with a K H / K D of about 3—4. The isotope effects were essentially unchanged when the reactions were partially inhibited with carbon monoxide. 2 None of the hydroxylations was significantly inhibited when NADPH was substituted with [4A‐ 2 H]‐ or [4B‐ 2 H]‐labeled NADPH. 3 When the reactions were carried out in deuterated water, there was no significant inhibition of 24‐hydroxylation of 5β‐cholestane‐3α,7α,12α‐triol, whereas 16α‐hydroxylation of pregnenolone was inhibited by 15%, 6β‐hydroxylation of testosterone by 30% and 26‐hydroxylation of 5β‐cholestane‐3α,7α,12α‐triol by 60%. As splitting of a C‐ 2 H bond in the rate‐limiting step is expected to decrease the rate of the overall reaction to less than one‐half, it was concluded that the rate‐limiting step in the 16α‐hydroxylation of pregnenolone and 24‐hydroxylation of 5β‐cholestane‐3α,7α,12α‐triol is the splitting of the C‐H bond in the substrate. Some other step is rate‐limiting in the 6β‐hydroxylation of testosterone and the 26‐hydroxylation of 5β‐cholestane‐3α,7α,12α‐triol. In the case of 26‐hydroxylation of 5β‐cholestane‐3α,7α,12α‐triol, water might participate in the rate‐limiting step by hydration or protonolysis of the enzyme. It is suggested that a common feature of hydroxylation in which splitting of the C‐H bond in the substrate is rate‐limiting is a relatively low sensitivity towards carbon monoxide.