tailieunhanh - Báo cáo khoa học: 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

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast ofChlamydomonas reinhardtiiis 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 residuesK128 andR197 had aKmfor NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. | Eur. J. Biochem. 271 4737-4744 2004 FEBS 2004 doi 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 Emmanuelle Graciet1 Guillermo Mulliert2 Sandrine Lebreton1 and Brigitte Gontero1 1Laboratoire Génétique et Membranes Departement Biologie Cellulaire Institut Jacques Monod UMR 7592 CNRS Universités Paris VI-VII Paris 2Laboratoire de cristallographie et de modelisation des materiaux mineraux et biologiques UMR 7036 Faculte des Sciences et Techniques Vandoeuvre-les-Nancy France 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 Km 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 .