Department of Molecular Biology and Ecology of Plants, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
J Exp Bot. 2011 Aug;62(12):4173-82. doi: 10.1093/jxb/err116. Epub 2011 May 6.
Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (K(cat)) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40-50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced K(cat) so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (P(max)) under saturating irradiance and Ci concentrations indicate that a >60% reduction in K(cat) can be tolerated before P(max) in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH.
正磷酸盐（Pi）刺激核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）的激活，而同时又抑制其催化作用。在三个 Pi 结合位点中，已经记录了 5P-和闩锁位点的作用，而 1P-位点的作用仍不清楚。取代了来自集胞藻 PCC6803 的 Rubisco 中 1P-位点的保守残基，并检查了酶衍生物的动力学特性以及对细胞光合作用和生长的影响。对于酶突变体 T65A/S 和 G404A，Pi 刺激的 Rubisco 激活减少，而 Pi 对催化的抑制作用保持不变。结合以前的研究结果，表明所有三个 Pi 结合位点都参与了 Rubisco 激活的刺激，而只有 5P-位点参与了催化的抑制。虽然所有突变都将 Rubisco 的催化周转率（Kcat）降低了 6-20 倍，但在饱和光照和无机碳（Ci）浓度下的光合作用和生长速率仅降低了 40-50%（在 T65A/S 突变体中）或根本没有降低（G404A 突变体）。对突变细胞的分析表明，Rubisco 含量增加了 3 倍，部分补偿了 Kcat 的降低，使得每叶绿素的羧化速率是野生型的三分之一。Rubisco 的动力学特性与饱和光照和 Ci 浓度下的光合速率（Pmax）之间的相关性表明，在 Synechocystsis PCC6803 的 Pmax 受到影响之前，可以容忍 Kcat 降低 60%以上。这些结果表明，Rubisco 活性对 Synechocystsis 光合作用速率的限制很低。卡尔文循环代谢物的测定表明，与高等植物不同，蓝细菌光合作用受到磷酸甘油酸还原的限制，可能是由于 ATP 或 NADPH 的限制。
