Ribulose 1,5-bisphosphate carboxylase: fate of the tritium label in [3-3H]ribulose 1,5-bisphosphate during the enzyme-catalyzed reaction

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Abstract
The reaction of [3-3H]ribulose 1,5-biphosphate and CO2 with [Rhodospirillum rubrum] ribulose-1,5-biphosphate carboxylase was investigated in order to provide information about the early steps of the enzyme-catalyzed reaction. The specific radioactivity of ribulose 1,5-biphosphate 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 H 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 for the transition states in the catalyzed reaction. It is evident that there is a rather fine balance among the 3 critical transition states for this reaction (those of enolization, condensation of the enediol with CO2, and solvent exchange of the C-3 proton).
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