Akhtar M, Calder M, Smith T, Wright J N
Biochem J. 1980 Feb 1;185(2):411-21. doi: 10.1042/bj1850411.
The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.
利用从足月人胎盘中获得的微粒体部分中存在的膜结合3-氧代甾体Δ4-Δ5-异构酶(EC 5.3.3.1)和3β-羟基甾体脱氢酶,研究了Δ5-3-氧代甾体异构化为Δ4-3-氧代甾体的机制。(1)描述了制备在4α-、4β-或6-位特异性标记的雄甾-5-烯-3β,17β-二醇的方法。(2)用在4α-或4β-位立体特异性3H标记的雄甾-5-烯-3β,17β-二醇进行孵育,结果表明异构化反应是通过立体特异性消除4β氢原子发生的。此外,从[4α-3H]雄甾-5-烯-3β,17β-二醇获得的Δ4-3-氧代甾体中3H的完全保留表明这些实验中非酶促作用可忽略不计。(3)为了研究进入的质子在C-6位插入的立体化学,从[6-3H]雄甾-5-烯-3β,17β-二醇的氧化异构化获得的[6-3H]雄甾-4-烯-3,17-二酮在6β位被匍枝根霉酶促羟基化。分离得到的6β-羟基雄甾-4-烯-3,17-二酮在6α位保留3H,这表明在异构酶催化的C(5)=C(6)双键迁移中,来自酶上酸性基团的进入质子必须从β面进入C-6,迫使现有的3H进入6α位。