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    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


    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

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    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:

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