Characterization of native and recombinant A4 glyceraldehyde 3-phosphate dehydrogenase. Kinetic evidence for confromation changes upon association with the small protein CP12.
A4 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purified from the green alga Chlamydomonas reinhardtii and was 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 pKs of 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.
Graciet
Emmanuelle
E
Institut Jacques Monod, Universités Paris VI-VII, Paris, France.
Lebreton
Sandrine
S
Camadro
Jean-Michel
JM
Gontero
Brigitte
B
D000818:Animals; D001426:Bacterial Proteins; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics; D019281:Dimerization; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D024602:Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins; D009249:NADP / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004163:Diphosphoglyceric Acids; C080820:MalD protein, Streptococcus pneumoniae; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C015891:glycerate 1,3-biphosphate; D009249:NADP; D024602:Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating)