Substrate‐Binding Isotherms of Spinach Chloroplastic Fructose‐1,6‐bisphosphatase and the Photoregulation of the Calvin Cycle

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Abstract
Fluorescence titration experiments of chloroplastic fructose-1,6-bisphosphatase by fructose bisphosphate and Mg were effected using the inactive dimeric, the inactive tetrameric and the active tetrameric enzyme forms. Mg binding to the inactive dimeric enzyme exhibits a positive cooperativity; fructose 1,6-bisphosphate exhibits no cooperativity at all. The binding of either Mg 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. Mg and fructose bisphosphate are bound to the active enzyme with a positive cooperativity. Non-linear least-square fitting allows one to estimate the binding constants and the free energy of binding of either Mg 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 Mg for the active or inactive enzyme forms are much greater than those of fructose bisphosphate. The metal ion gives to the enzyme the right conformation to bind the sugar PO4.