Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde‐3‐phosphate dehydrogenase in the assembly process of a bi‐enzyme complex involved in CO 2 assimilation

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.