Substrate-binding isotherms of spinach chloroplastic fructose-1,6-bisphosphatase and the photoregulation of the Calvin cycle.

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.