Saver B G, Knowles J R
Biochemistry. 1982 Oct 26;21(22):5398-403. doi: 10.1021/bi00265a003.
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.
当在由深红红螺菌的核酮糖-1,5-二磷酸羧化酶催化的羧化反应中，让核酮糖1,5-二磷酸与氚化水中的二氧化碳反应时，发现部分反应后重新分离出的核酮糖1,5-二磷酸带有放射性标记。在反应过程中，剩余底物池的比放射性增加。在氧化氘中的实验表明，同位素标记位于碳3上。早期未能检测到这种交换过程可能是由于使用了在没有二氧化碳时无活性的酶。目前的结果为核酮糖1,5-二磷酸的C-2和C-3之间烯二醇中间体的存在提供了直接证据，并表明烯醇化步骤在整个羧化酶反应中至少部分是限速的。产物3-磷酸-D-甘油酸的比放射性在整个反应过程中保持恒定，约为溶剂比放射性的六分之一。这加强了反对“粘性”质子参与反应的论点。
