Two phosphoenolpyruvate carboxykinases coexist in the Crassulacean Acid Metabolism plant Ananas comosus. Isolation and characterization of the smaller 65 kDa form

• Published in: Plant physiology and biochemistry 2011-06, Vol.49 (6), p.646-653
• Main Authors: Martín, Mariana, Rius, Sebastián Pablo, Podestá, Florencio Esteban
• Format: Article
• Language: English
• Subjects: Ananas - enzymology; Biological and medical sciences; Carbon metabolism; Crassulacean Acid Metabolism; Decarboxylation; Fundamental and applied biological sciences. Psychology; Oxaloacetic Acid - metabolism; Phosphoenolpyruvate - metabolism; Phosphoenolpyruvate carboxykinase; Phosphoenolpyruvate Carboxylase - chemistry; Phosphoenolpyruvate Carboxylase - isolation & purification; Photosynthesis - physiology; Pineapple; Plant Leaves - chemistry; Plant physiology and development; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Protein Isoforms; Proteolysis; Temperature
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/j.plaphy.2011.02.015

Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30 °C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase. ► Two PEPCK isoforms of 74 and 65 kDa were coexist in vivo in leaves of pineapple. ► Characterization of the 65-kDa PEPCK showed that it possess regulatory properties. ► The results suggest that it could participate in C and N metabolism as a decarboxylase.