Jordan D B, Chollet R
J Biol Chem. 1983 Nov 25;258(22):13752-8.
Substrate ribulose bisphosphate is a potent and a weak inhibitor of the rate of CO2/Mg2+ activation in the carboxylase purified from spinach leaves and Rhodospirillum rubrum, respectively. At 2 degrees C, the concentration of ribulose bisphosphate required for 50% inhibition of the initial rate of CO2/Mg2+ activation was less than 0.4 microM for the spinach enzyme, but between 67 and 270 microM for the R. rubrum carboxylase. Activator 14CO2 trapping experiments demonstrated that ribulose bisphosphate inhibits activation by excluding activator CO2 from the spinach enzyme. The reason for the different sensitivities to inhibition by substrate was evident from equilibrium binding studies with the inactive enzyme forms which indicated that the KD (ribulose bisphosphate) was 0.021 microM for spinach enzyme and 5.9 microM for the R. rubrum protein. Inhibition of activation, however, was not explained by the equilibrium binding results alone. Ribulose bisphosphate was observed to dissociate very slowly from the inactive spinach enzyme (at 2 degrees C, kOFF = 4.9 X 10(-5) s-1). The release of substrate from the inactive R. rubrum carboxylase was much more rapid, with a minimum value for kOFF estimated at 5 X 10(-3) s-1 at 2 degrees C. We conclude that strong inhibition of CO2/Mg2+ activation in the spinach enzyme is mediated by the tight binding and slow release of ribulose bisphosphate, which prevent activator CO2 and Mg2+ from binding to the protein. Weak inhibition of activation in the R. rubrum enzyme results from a larger KD value and a more rapid exchange of ribulose bisphosphate, which allow activator CO2 and Mg2+ to bind to the free enzyme between successive substrate-binding events.
底物核酮糖二磷酸分别是从菠菜叶和深红红螺菌中纯化得到的羧化酶中CO₂/Mg²⁺激活速率的强效和弱抑制剂。在2℃时，菠菜酶对CO₂/Mg²⁺激活初始速率产生50%抑制所需的核酮糖二磷酸浓度小于0.4微摩尔，而深红红螺菌羧化酶的该浓度在67至270微摩尔之间。活化剂¹⁴CO₂捕获实验表明，核酮糖二磷酸通过阻止活化剂CO₂与菠菜酶结合来抑制激活。从与无活性酶形式的平衡结合研究中可以明显看出对底物抑制敏感性不同的原因，该研究表明菠菜酶的KD（核酮糖二磷酸）为0.021微摩尔，深红红螺菌蛋白的KD为5.9微摩尔。然而，激活的抑制不能仅由平衡结合结果来解释。观察到核酮糖二磷酸从无活性的菠菜酶上解离非常缓慢（在2℃时，kOFF = 4.9×10⁻⁵ s⁻¹）。无活性的深红红螺菌羧化酶中底物的释放要快得多，在2℃时kOFF的最小值估计为5×10⁻³ s⁻¹。我们得出结论，菠菜酶中CO₂/Mg²⁺激活的强烈抑制是由核酮糖二磷酸的紧密结合和缓慢释放介导的，这阻止了活化剂CO₂和Mg²⁺与蛋白质结合。深红红螺菌酶中激活的弱抑制是由于较大的KD值和核酮糖二磷酸更快的交换，这使得活化剂CO₂和Mg²⁺能够在连续的底物结合事件之间与游离酶结合。
