Structural characterization and the determination of negative cooperativity in the tight binding of 2-carboxyarabinitol bisphosphate to higher plant ribulose bisphosphate carboxylase.
When CO2/Mg2+-activated spinach leaf ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) is incubated with the transition-state analog 2-carboxyarabinitol 1,5-bisphosphate, an essentially irreversible complex is formed. The extreme stability of this quaternary complex has allowed the use of native analytical isoelectric focusing, anion-exchange chromatography, and nondenaturing polyacrylamide gel electrophoresis to probe the mechanism of the binding process and the effects of ligand tight-binding on the structure of the protein molecule. Changes in the chromatographic and electrophoretic properties of the enzyme upon tight binding of the inhibitor reveal that the ligand induces a conformational reorganization which extends to the surface of the protein molecule and, at saturation, results in a 16% decrease in apparent molecular weight. Analysis of ligand binding by isoelectric focusing shows that (i) incubating the protein with a stoichiometric molar concentration of ligand (site basis) results in an apparently charge homogeneous enzyme population with an isoelectric point of 4.9, and (ii) substoichiometric levels of ligand produce differential effects on each of the charge microheterogeneous native enzyme forms. These stoichiometry-dependent changes in electrofocusing band patterns were employed as a probe of cooperativity in the ligand tight-binding process. The tight-binding reaction was shown to be negatively cooperative.
D002852:Chromatography, Ion Exchange; D003035:Cobalt / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D007525:Isoelectric Focusing; D008274:Magnesium / Q000378:metabolism; D008433:Mathematics; D010428:Pentosephosphates; D010944:Plants / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D013402:Sugar Alcohols; D013403:Sugar Phosphates / Q000378:metabolism*
D010428:Pentosephosphates; D013402:Sugar Alcohols; D013403:Sugar Phosphates; C032687:2-carboxyarabinitol 1,5-biphosphate; D003035:Cobalt; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium