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Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3‐phosphate dehydrogenase/CP12 complex – purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii
Author(s) -
Erales Jenny,
Avilan Luisana,
Lebreton Sandrine,
Gontero Brigitte
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06284.x
Subject(s) - aldolase a , fructose bisphosphate aldolase , chlamydomonas reinhardtii , aldolase b , dehydrogenase , dissociation constant , biochemistry , glyceraldehyde , phosphofructokinase 2 , fructose , biology , chemistry , enzyme , mutant , receptor , gene
Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii , were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3‐phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6‐bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 ± 0.05 μ m and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3‐phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55±5 n m . Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii , we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis–Menten kinetics with fructose 1,6‐bisphosphate and sedoheptulose 1,7‐bisphosphate, with a catalytic constant equal to 35 ± 1 s −1 and 4 ± 0.1 s −1 , respectively. The K m value for fructose 1,6‐bisphosphate was equal to 0.16 ± 0.02 m m and 0.046 ± 0.005 m m for sedoheptulose 1,7‐bisphosphate. The catalytic efficiency of aldolase was thus 219 ± 31 s −1 ·m m −1 with fructose 1,6‐bisphosphate and 87 ± 9 s −1 ·m m −1 with sedoheptulose 1,7‐bisphosphate. In the presence of the complex, this parameter for fructose 1,6‐bisphosphate increased to 310 ± 23 s −1 ·m m −1 , whereas no change was observed with sedoheptulose 1,7‐bisphosphate. The condensation reaction of aldolase to form fructose 1,6‐bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.