The Interaction of Metal Ions with Ribulose 1,5-Diphosphate Carboxylase from Spinach

Abstract

Abstract 2-Carboxy-d-ribitol 1,5-diphosphate, an analogue of the proposed six-carbon intermediate in the reaction catalyzed by d-ribulose 1,5-diphosphate carboxylase, is a potent inhibitor of this enzyme. Tight binding of 2-carboxy-d-ribitol 1,5-diphosphate to d-ribulose 1,5-diphosphate carboxylase is observed only in the presence of divalent metal ions, e.g. Mn2+, Mg2+. The complex formed contains equimolar amounts of inhibitor and divalent metal ion when isolated by gel filtration. The extent of tight binding observed is dependent on the time of preliminary incubation prior to filtration. A similar increase in the extent of tight binding is observed on prolonged incubation of d-ribulose 1,5-diphosphate carboxylase with 14C-d-ribulose 1,5-diphosphate, Mg2+ (or Mn2+), KHCO3, glutathione, and cyanide. The complex formed contains equimolar amounts of 14C-d-ribulose 1,5-diphosphate and divalent metal ion. In several instances the maximal extent of tight binding of 2-carboxy-d-ribitol 1,5-diphosphate and of d-ribulose 1,5-diphosphate (in the presence of cyanide) as estimated by gel filtration approximates the total number of binding sites for these ligands as estimated by ultrafiltration. These studies suggest a role for the divalent metal ion in formation or stabilization of the six-carbon intermediate which may result from condensation of d-ribulose 1,5-diphosphate and CO2. The bound Cu2+ present in d-ribulose 1,5-diphosphate carboxylase has been removed by incubation with cyanide. The copper-free enzyme readily rebinds Cu2+. No significant kinetic or structural differences are observed between the native and the copper-free enzymes. Both enzyme species form the enzyme-d-ribulose 1,5-diphosphate-cyanide ternary complex (Wishnick, M., and Lane, M. D., J. Biol. Chem., 244, 55 (1969)). Important differences are, however, observed in the extent and properties of the tight binding of 2-carboxy-d-ribitol 1,5-diphosphate and of d-ribulose 1,5-diphosphate plus cyanide. Divalent metal ion is not required for binding of 2-carboxyribitol 1,5-diphosphate to the copper-free enzyme and, in most instances, the extent of binding of these ligands is greater for the copper-free as compared with the native enzyme. These studies suggest that the bound copper is involved in maintenance of a particular conformation of d-ribulose 1,5-diphosphate carboxylase.