TAILIEUCHUNG - Báo cáo khoa học: Characterization of native and recombinant A4 glyceraldehyde 3-phosphate dehydrogenase Kinetic evidence for conformation changes upon association with the small protein CP12

A4 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purified from the green algaChlamydomonas reinhardtii andwas alsooverexpressed inEscherichia coli. Bothpurified A4 tetramers of recombinant and native GAPDH were characterized for the first time. The pH optimum for both nativeandrecombinant enzymeswas close theresidues involvedincatalysis indicate thatacysteineanda histidinemay take part in catalysis by chloroplast GAPDH, as is the case for glycolytic GAPDH. | Eur. J. Biochem. 270 129-136 2003 FEBS 2003 doi Characterization of native and recombinant A4 glyceraldehyde 3-phosphate dehydrogenase Kinetic evidence for conformation changes upon association with the small protein CP12 Emmanuelle Graciet Sandrine Lebreton Jean-Michel Camadro and Brigitte Gontero Institut Jacques Monod Universites Paris VI-VII Paris France A glyceraldehyde 3-phosphate dehydrogenase GAPDH was purified from the green alga Chlamydomonas reinhardtii and was also overexpressed in Escherichia coli. Both purified A tetramers of recombinant and native GAPDH were characterized for the first time. The pH optimum for both native and recombinant enzymes was close to . 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. Keywords GAPDH CP12 overexpression purification kinetics. The enzyme glyceraldehyde 3-phosphate dehydrogenase GAPDH exists as two main forms in higher plants and algae. The cytosolic form is involved in glycolysis while the chloroplast form is involved in the .

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