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

Abstract

When ribulose 1,5-bisphosphate is allowed to react with CO2 in tritiated water in the carboxylation reaction catalyzed by ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, the ribulose 1,5-bisphosphate reisolated from partial reaction is 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 C-3. Earlier failures to detect this exchange process probably derive from the use of enzyme that is, in the absence of CO2, 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.