Yokota A, Tsujimoto N
Department of Agricultural Chemistry, University of Osaka Prefecture, Japan.
Eur J Biochem. 1992 Mar 1;204(2):901-9. doi: 10.1111/j.1432-1033.1992.tb16710.x.
Ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO] from plant sources shows a biphasic reaction course when assayed with more than 2 mM ribulose 1,5-bisphosphate [Rbu(1,5)P2]. In the burst, Rbu(1,5)P2CO has its substrate-binding sites occupied with Rbu(1,5)P2 for the initial few minutes, then both substrate-binding and regulatory sites are occupied by Rbu(1,5)P2 in the subsequent linear phase, at physiological concentrations of Rbu(1,5)P2 [A. Yokota (1991) J. Biochem. (Tokyo) 110, 246-252]. This study attempts the characterization of spinach Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the regulatory sites and the interaction of Rbu(1,5)P2CO activase with Rbu(1,5)P2CO purified with poly(ethylene glycol) 4000 without denaturation. Binding of Rbu(1,5)P2 to the regulatory sites strongly influences the temperature dependence of the carboxylase activity of Rbu(1,5)P2CO. The activation energy of Rbu(1,5)P2CO with Rbu(1,5)P2 at the regulatory sites was 40% larger than that without Rbu(1,5)P2 over 30 degrees C, although the binding did not affect the activation energy below this temperature. This caused the almost linear reaction course of the carboxylase reaction at 50 degrees C. The optimum pH for the activity of Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the sites was 8.0-8.2, and increased by about pH 0.2 from that of Rbu(1,5)P2CO without Rbu(1,5)P2. The ratio of the activity of the former form to that of the latter increased with increasing pH with an inflection point at pH 8.1. The increase in the ratio was accompanied by a decrease in the hysteric conformational change of Rbu(1,5)P2CO. The ATP-hydrolyzing activity inherent to Rbu(1,5)P2CO activase was stimulated about twofold by 3-5 mM Rbu(1,5)P2. Rbu(1,5)P2CO in the inactive complex with Rbu(1,5)P2 experienced hysteresis and bound Rbu(1,5)P2 at the regulatory sites during activation in the presence of Rbu(1,5)P2CO activase. Evidence was obtained that Rbu(1,5)P2CO activase promoted the activation of Rbu(1,5)P2CO through binding to the large subunits of Rbu(1,5)P2CO.
当用超过2 mM的1,5 - 二磷酸核酮糖[Rbu(1,5)P2]进行测定时，来自植物源的1,5 - 二磷酸核酮糖羧化酶/加氧酶[Rbu(1,5)P2CO]呈现出双相反应过程。在初始阶段，Rbu(1,5)P2CO的底物结合位点在最初几分钟被Rbu(1,5)P2占据，然后在随后的线性阶段，在生理浓度的Rbu(1,5)P2下，底物结合位点和调节位点都被Rbu(1,5)P2占据[A. Yokota(1991) J. Biochem. (Tokyo) 110, 246 - 252]。本研究试图对在调节位点携带Rbu(1,5)P2的菠菜Rbu(1,5)P2CO进行表征，并研究Rbu(1,5)P2CO活化酶与用聚乙二醇4000纯化且未变性的Rbu(1,5)P2CO之间的相互作用。Rbu(1,5)P2与调节位点的结合强烈影响Rbu(1,5)P2CO羧化酶活性的温度依赖性。在30℃以上，调节位点带有Rbu(1,5)P2的Rbu(1,5)P2CO的活化能比没有Rbu(1,5)P2时大40%，尽管这种结合在该温度以下不影响活化能。这导致羧化酶反应在50℃时几乎呈线性反应过程。在调节位点携带Rbu(1,5)P2的Rbu(1,5)P2CO活性的最适pH为8.0 - 8.2，比没有Rbu(1,5)P2的Rbu(1,5)P2CO的最适pH高约0.2。前一种形式与后一种形式的活性比随pH升高而增加，在pH 8.1处有一个拐点。该比值的增加伴随着Rbu(1,5)P2CO滞后构象变化的减少。Rbu(1,5)P2CO活化酶固有的ATP水解活性被3 - 5 mM的Rbu(1,5)P2刺激约两倍。与Rbu(1,5)P2形成无活性复合物的Rbu(1,5)P2CO在Rbu(1,5)P2CO活化酶存在下活化过程中经历滞后现象，并在调节位点结合Rbu(1,5)P2。有证据表明，Rbu(1,5)P2CO活化酶通过与Rbu(1,5)P2CO的大亚基结合促进Rbu(1,5)P2CO的活化。
