RATE-LIMITING STEP IN MICROSOMAL HYDROXYLATION OF STEROIDS
RATE-LIMITING STEP IN MICROSOMAL HYDROXYLATION OF STEROIDS
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DOI：
10.1111/j.1432-1033.1972.tb01845.x
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发表时间：
1972-01-01
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影响因子：
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通讯作者：
BJORKHEM, I
中科院分区：
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作者：
BJORKHEM, I
研究了5β-胆甾烷-3 α，7 α，12 α-三醇的微粒体NADPH依赖性24-和26-羟基化、睾酮的6β-羟基化和双烯醇酮的16α-羟基化中的限速步骤。当底物中的氢在羟基化过程中被去除，被氘取代时，当NADPH中的4A-或4 B-氢被氘取代时，当在氘化水中进行孵育时，[6β-2H，1，2 - 3 H2]-加[4 - 14 C]标记睾酮的16 β-羟基化和5β-[26 - 2 H3]-胆甾烷-3 α，7 α的26-羟基化，12α-三醇不涉及显著的同位素效应，而[16α-2H，3α-3H]-加[4 - 14 C]-标记的阿替诺龙的16α-羟基化和5β-[24 - 2 H2]胆甾烷-3 α，7 α，12 α-三醇的24-羟基化涉及同位素效应，aKH/KD约为3-4。当反应被一氧化碳部分抑制时，同位素效应基本上没有变化。2当NADPH被[4A-2 H]-或[4 B-2 H]-标记的NADPH取代时，羟基化没有受到显著抑制。3当反应在氘代水中进行时，5β-胆甾烷-3 α，7 α，12 α-三醇的24-羟基化没有受到显著抑制，而15%的去甲烯醇酮的16α-羟基化、30%的睾酮的6β-羟基化和5β-胆甾烷-3 α，7 α的26-羟基化抑制了去甲烯醇酮的16α-羟基化，由于在限速步骤中C-2 H键的分裂预期将总反应速率降低至小于一半，可以得出结论，在双烯醇酮的16α-羟基化和5β-胆甾烷-3 α，7 α，12 α-三醇的24-羟基化中的限速步骤是底物中C-H键的裂解。在睾酮的6β-羟基化和5β-胆甾烷-3 α，7 α，12 α-三醇的26-羟基化中，其他一些步骤是限速的。在5β-胆甾烷-3 α，7 α，12 α-三醇26-羟基化的情况下，水可能通过酶的水合或质子分解参与限速步骤。这表明，其中底物中C-H键的分裂是速率限制的羟基化的共同特征是对一氧化碳的敏感性相对较低。
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.16β‐Hydroxylation of [6β‐2H, 1,2‐3H2]‐plus [4‐14C]labeled testosterone and 26‐hydroxylation of 5β‐[26‐2H3]‐cholestane‐3α,7α,12α‐triol did not involve significant isotope effect, whereas 16α‐hydroxylation of [16α‐2H, 3α‐3H]‐plus [4‐14C]‐labeled pregnenolone and 24‐hydroxylation of 5β‐[24‐2H2]cholestane‐3α,7α,12α‐triol involved isotope effects with aKH/KDof about 3—4. The isotope effects were essentially unchanged when the reactions were partially inhibited with carbon monoxide.2None of the hydroxylations was significantly inhibited when NADPH was substituted with [4A‐2H]‐ or [4B‐2H]‐labeled NADPH.3When 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‐2H 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.