Purohit U S, Verma N C, Bhagwat A S
Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, Bombay, India.
FEBS Lett. 1991 Jun 17;284(1):23-6. doi: 10.1016/0014-5793(91)80752-o.
The spinach ribulose 1,5-bisphosphate carboxylase/oxygenase was labelled with o-phthalaldehyde, which forms a stable fluorescent isoindole adduct at the active site. The fluorescence behaviour of the labelled enzyme after activation to different levels by Mg2+ was compared with that of a synthetic isoindole adduct of o-phthalaldehyde, namely 1-(hydroxyethylthio)-2-beta hydroxyethylisoindole in solvents of different pH and polarity. The results suggest that the microenvironment at the catalytically incompetent active site of the unactivated Rubisco is highly acidic (pH less than 2) in nature. The activation by Mg2+ results in the conformational change such that the effective pH at the active site increases to greater than 8. The polarity of the active site of the activated enzyme was found to be similar to that of a mixture of hexane and toluene.
菠菜核酮糖-1,5-二磷酸羧化酶/加氧酶用邻苯二甲醛进行标记，邻苯二甲醛在活性位点形成稳定的荧光异吲哚加合物。将经Mg2+激活至不同水平后的标记酶的荧光行为，与邻苯二甲醛的一种合成异吲哚加合物，即1-(羟乙硫基)-2-β-羟乙异吲哚在不同pH和极性溶剂中的荧光行为进行比较。结果表明，未活化的核酮糖-1,5-二磷酸羧化酶催化无活性的活性位点处的微环境本质上是高酸性的（pH小于2）。Mg2+激活导致构象变化，使得活性位点处的有效pH增加到大于8。发现活化酶活性位点的极性与己烷和甲苯混合物的极性相似。
