Sue J M, Knowles J R
Biochemistry. 1982 Oct 26;21(22):5404-10. doi: 10.1021/bi00265a004.
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).
为了获取有关该酶催化反应早期步骤的信息，人们对[3-³H]核酮糖1,5-二磷酸与二氧化碳和核酮糖-1,5-二磷酸羧化酶的反应进行了研究。部分反应后重新分离得到的核酮糖1,5-二磷酸的比放射性随着反应的进行而升高，这表明同位素歧视（导致未标记底物的优先消耗）比C-3上的氢与溶剂质子的平衡更为重要。这些数据证实了烯二醇中间体的存在，并为允许的自由能水平范围设定了限制，即该反应的三个关键过渡态（烯醇化、烯二醇与二氧化碳的缩合以及C-3质子的溶剂交换）之间存在相当精细的平衡。
