Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme complex involved in CO2 assimilation
Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme complex involved in CO2 assimilation
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DOI：
10.1111/j.1432-1033.2004.04437.x
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发表时间：
2004-12-01
期刊：
影响因子：
--
通讯作者：
Gontero, B
中科院分区：
文献类型：
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作者：
Graciet, E;Mulliert, G;Gontero, B
莱茵衣藻叶绿体中的甘油醛-3-磷酸脱氢酶（GAPDH）是一种复合物的一部分，该复合物还包括磷酸核酮糖激酶（phosphoribulokinase，CP 12）。我们通过将K128和R197残基改变为A或E，确定了该复合物中参与蛋白质-蛋白质相互作用的两个GAPDH残基。K128 A/E突变体对NADH的Km是野生型的两倍，并且无论辅因子如何，催化常数都较低。突变体R197 A的动力学与野生型相似，而R197 E突变体与NADPH的催化常数较低。只有突变附近的小的结构变化可能导致这些差异，因为圆二色性和荧光光谱与野生型GAPDH相似。对突变体的分子建模得出了同样的结论。除R197 E外，所有突变体均重构GAPDH-CP 12亚复合物。尽管通过表面等离子体共振测量的解离常数在突变体中比野生型GAPDH和CP 12高10-70倍，但它们仍然很低。对于R197 E突变，我们计算了4 kcal/mol的去稳定作用，这可能对应于GAPDH和CP 12之间相互作用的盐桥的稳定作用的丧失。所有的突变体GAPDH-CP 12亚复合物不能与β-内酰胺酶相互作用并形成天然复合物。与野生型GAPDH-CP 12相比，所有突变体GAPDH-CP 12亚复合物的动力学变化的缺乏表明突变体不经历对CAPDH-CP 12结合必需的构象变化。
The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast of Chlamydomonas reinhardtii is part of a complex that also includes phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved in protein-protein interactions in this complex, by changing residues K128 and R197 into A or E. K128A/E mutants had a K-m for NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. The kinetics of the mutant R197A were similar to those of the wild type, while the R197E mutant had a lower catalytic constant with NADPH. Only small structural changes near the mutation may have caused these differences, since circular dichroism and fluorescence spectra were similar to those of wild-type GAPDH. Molecular modelling of the mutants led to the same conclusion. All mutants, except R197E, reconstituted the GAPDH-CP12 subcomplex. Although the dissociation constants measured by surface plasmon resonance were 10-70-fold higher with the mutants than with wild-type GAPDH and CP12, they remained low. For the R197E mutation, we calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of the stabilizing effect of a salt bridge for the interaction between GAPDH and CP12. All the mutant GAPDH-CP12 subcomplexes failed to interact with PRK and to form the native complex. The absence of kinetic changes of all the mutant GAPDH-CP12 subcomplexes, compared to wild-type GAPDH-CP12, suggests that mutants do not undergo the conformation change essential for PRK binding.