Ribulose-1,5-bisphosphate carboxylase: enzyme-catalyzed appearance of solvent tritium at carbon 3 of ribulose 1,5-bisphosphate reisolated after partial reaction.

When ribulose 1,5-bisphosphate is allowed to react with carbon dioxide in tritiated water in the carboxylation reaction catalyzed by ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, the ribulose 1,5-bisphosphate reisolated after partial reaction is found to be labeled. The specific radioactivity of the remaining substrate pool rises during the course of the reaction. Experiments in deuterium oxide show that the isotopic label resides on carbon 3. Earlier failures to detect this exchange process probably derive from the use of enzyme that was, in the absence of carbon dioxide, inactive. The present results provide direct evidence for the intermediacy of the enediol between C-2 and C-3 of ribulose 1,5-bisphosphate and show that the enolization step is at least partially rate limiting in the overall carboxylase reaction. The specific radioactivity of the product 3-phospho-D-glycerate remains constant throughout the course of the reaction at about one-sixth that of the solvent. This strengthens the argument against the involvement of "sticky" protons in the reaction.