Graciet, Emmanuelle 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

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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:	http://onlinelibrary.wiley.com
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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