Ribulose-1,5-bisphosphate Carboxylase: Fate of the Tritium Label in [3]3H]ribulose 1,5-bisphosphate During the Enzyme-catalyzed Reaction

The reaction of [3-3H]ribulose 1,5-bisphosphate and CO2 with ribulose-1,5-bisphosphate carboxylase has been investigated in order to provide information about the early steps of the enzyme-catalyzed reaction. The specific radioactivity of ribulose 1,5-bisphosphate reisolated after partial reaction rises as the reaction proceeds, demonstrating that the isotopic discrimination (which results in the preferential consumption of unlabeled substrate) is more important than the equilibration of the hydrogen on C-3 with solvent protons. These data confirm the existence of the enediol intermediate and set limits on the range of permissible free-energy levels that there is a rather fine balance among the three critical transition states for this reaction (those of enolization, condensation of the enediol with CO2, and solvent exchange of the C-3 proton).