Meunier J C, Buc J, Soulié J M, Pradel J, Ricard J
Eur J Biochem. 1981 Jan;113(3):513-20. doi: 10.1111/j.1432-1033.1981.tb05093.x.
Fluorescence titration experiments of chloroplastic fructose-1,6-bisphosphatase by fructose bisphosphate and magnesium have been effected using the inactive dimeric, the inactive tetrameric and the active tetrameric enzyme forms. Magnesium binding to the inactive dimeric enzyme exhibits a positive cooperativity whereas fructose 1,6-bisphosphate exhibits no cooperativity at all. The binding of either magnesium or fructose bisphosphate to the inactive oxidized tetramer exhibits a succession of negative and positive cooperativities (mixed cooperativity). Upon reduction of the inactive tetramer by dithiothreitol and activation, the ligand binding properties of the enzyme are changed. Magnesium and fructose bisphosphate are bound to the active enzyme with a positive cooperativity. Non-linear least-square fitting allows one to estimate thea binding constants, and therefore the free energy of binding of either magnesium or fructose bisphosphate to the various forms of the enzyme. Whatever the state of fructose-1,6-bisphosphatase, one ligand is bound per subunit. The affinity constants of magnesium for the active or inactive enzyme forms are much greater than those of fructose bisphosphate. This suggests that the metal ion gives to the enzyme the right conformation to bind the sugar phosphate.
利用无活性二聚体、无活性四聚体和活性四聚体形式的叶绿体果糖-1,6-二磷酸酶，进行了果糖二磷酸和镁对其的荧光滴定实验。镁与无活性二聚体酶的结合表现出正协同性，而果糖1,6-二磷酸则完全没有协同性。镁或果糖二磷酸与无活性氧化四聚体的结合表现出一系列负协同性和正协同性（混合协同性）。用二硫苏糖醇还原无活性四聚体并激活后，酶的配体结合特性发生改变。镁和果糖二磷酸以正协同性与活性酶结合。非线性最小二乘法拟合可以估算结合常数，从而估算镁或果糖二磷酸与各种形式酶结合的自由能。无论果糖-1,6-二磷酸酶处于何种状态，每个亚基结合一个配体。镁对活性或无活性酶形式的亲和常数远大于果糖二磷酸的亲和常数。这表明金属离子赋予酶正确的构象以结合磷酸糖。
