Biochemical characterization of phosphoenolpyruvate carboxykinases from Arabidopsis thaliana

ROJAS, BRUNO E.; HARTMAN, MATÍAS D.; FIGUEROA, CARLOS M.; LEADEN, LAURA; PODESTÁ, FLORENCIO E.; IGLESIAS, ALBERTO A.

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Lugar: Londres; Año: 2019 vol. 476 p. 2939 - 2952

0264-6021

ATP-dependent phosphoenolpyruvatecarboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants havebeen widely studied due to their crucial role in photosynthetic CO2 fixation.However, our knowledge on the structural, kinetic and regulatory properties ofthe enzymes from C3 species is still limited. In this work, wereport the recombinant production and biochemical characterization of twoPEPCKs identified in Arabidopsis thaliana:AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetateand ATP, reinforcing their role as decarboxylases. We employed ahigh-throughput screening for putative allosteric regulators using differentialscanning fluorometry and confirmed their effect on enzyme activity byperforming enzyme kinetics. AthPEPCK1 and AthPEPCK2 areallosterically modulated by key intermediates of plant metabolism, namelysuccinate, fumarate, citrate and α-ketoglutarate. Interestingly,malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had noeffect on AthPEPCK2. Overall, our results demonstrate that the enzymesinvolved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fineallosteric regulation.