Purification and characterization of pineapple leaf phosphoenolpyruvate carboxykinase (PEPCK)

MARTÍN, M; RIUS, SP; PODESTÁ, F

Villa Carlos Paz-Córdoba

Congreso; XXXVIII Reunión Nacional. Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2002

SAIB

In PEPCK-type CAM plants, a futile cycle is possible between this enzyme and phosphoenolpyruvate carboxylase (PEPC). Unlike PEPC, little is know about PEPCK regulation. To address this issue, pineapple leaf was purified 108-fold to a maximal activity of 166.68 U/mg (4% yield). Decarboxilating activity was maximal at pH 7.7  and increased with temperature up to 32°C, while on the carboxilation direction (optimal pH 7.3) the activity increased up to 40°C. Biphasic Arrenhius plots showed a transition temperature of 27.3 or 30.3°C for decarboxilation and carboxilation, respectively. Km values for substrates were: oxalacetate, 42 uM; ATP, 0.12 mM and phosphoenolpyruvate, 0.56  mM. ADP bound in a cooperative fashion to PEPCK with an S0.5 of 47 uM (nh= 2). Succinate activated decarboxilation, while citrate, L-malate and 3-PGA inhibited the carboxilating activity. The purified enzyme showed a subunit mass of 64 kDa. Antibodies raised against this polypeptide revealed bands of 78 and 60 kDa in creude extracts, hinting that truncated, albeit active, enzyme has been purified. Overall kinetic properties unveiled for PEPCK indicates that it is more efficient in the decarboxylating direction. Efforts are being directed to purifying the intact PEPCKand compare its propierties with the truncated enzyme. Supported by grants from ANPCyT and Fundación Antorchas.