MURAL - Maynooth University Research Archive Library



    Characterization of native and recombinant A4 glyceraldehyde 3-phosphate dehydrogenase. Kinetic evidence for conformation changes upon association with the small protein CP12


    Graciet, Emanuelle and Lebreton, Sandrine and Camadro, Jean-Michel and Gontero, Brigitte (2003) Characterization of native and recombinant A4 glyceraldehyde 3-phosphate dehydrogenase. Kinetic evidence for conformation changes upon association with the small protein CP12. European Journal of Biochemistry, 270 (1). pp. 129-136. ISSN 0014-2956

    [img]
    Preview
    Download (207kB) | Preview


    Share your research

    Twitter Facebook LinkedIn GooglePlus Email more...



    Add this article to your Mendeley library


    Abstract

    A4 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purified from the green alga Chlamydomonas reinhardtii andwas also overexpressed in Escherichia coli. Both purified A4 tetramers of recombinant and native GAPDH were characterized for the first time. The pH optimum for both native and recombinant enzymes was close to 7.8. The pKsof the residues involved in catalysis indicate that a cysteine and a histidine may take part in catalysis by chloroplast GAPDH, as is the case for glycolytic GAPDH. Native and recombinant GAPDH show Michaelis–Menten kinetics with respect to their cofactors, NADH and NADPH, with greater specificity for NADPH. The kinetic parameters are similar to those of the heterotetrameric A2B2 spinach chloroplast GAPDH. Native C. reinhardtii and recombinant GAPDHs exhibit a cooperative behavior towards the substrate 1,3-bisphosphoglycerate (BPGA). This positive cooperativity is specific to the C. reinhardtii enzyme, as higher plant A2B2 GAPDHs show Michaelis–Menten kinetics. Native GAPDH has twofold lower catalytic constant and K0.5 for BPGA than recombinant GAPDH. Mass spectrometry analysis of native GAPDH shows that it is a complex of GAPDH and the small protein CP12. In vitro reconstitution assays indicate that the kinetic differences are the result conformation changes of GAPDH upon association with CP12.

    Item Type: Article
    Keywords: GAPDH; CP12; overexpression; purification; kinetics;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 7438
    Identification Number: https://doi.org/10.1046/j.1432-1033.2003.03372.x
    Depositing User: Emanuelle Graciet
    Date Deposited: 09 Sep 2016 15:14
    Journal or Publication Title: European Journal of Biochemistry
    Publisher: Wiley-Blackwell
    Refereed: Yes
    URI:

    Repository Staff Only(login required)

    View Item Item control page

    Downloads

    Downloads per month over past year