Graciet, Emmanuelle and Mulliert, Guillermo and Lebreton, Sandrine and Gontero, Brigitte
(2004)
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.
European Journal of Biochemistry, 271 (23-24).
pp. 4737-4744.
ISSN 0014-2956
Abstract
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 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.
| Item Type: |
Article
|
| Keywords: |
phosphoribulokinase; glyceraldehyde-3-phosphate
dehydrogenase; CP12; site-directed mutagenesis;
protein–protein interactions; |
| Academic Unit: |
Faculty of Science and Engineering > Biology |
| Item ID: |
7433 |
| Identification Number: |
https://doi.org/10.1111/j.1432-1033.2004.04437.x |
| Depositing User: |
Emanuelle Graciet
|
| Date Deposited: |
06 Sep 2016 15:44 |
| Journal or Publication Title: |
European Journal of Biochemistry |
| Publisher: |
Wiley-Blackwell |
| Refereed: |
Yes |
| URI: |
|
| Use Licence: |
This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available
here |
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