title: Two phosphoenolpyruvate carboxykinases coexist in the Crassulacean Acid Metabolism plant Ananas comosus. Isolation and characterization of the smaller 65 kDa form.
abstract: 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.
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
authors.forename: Mariana
authors.initials: M
authors.lastname: Martín
authors.forename: Sebastián Pablo
authors.initials: SP
authors.lastname: Rius
authors.forename: Florencio Esteban
authors.initials: FE
authors.lastname: Podestá
mesh_terms: D031230:Ananas / Q000201:enzymology*; D003653:Decarboxylation; D062907:Oxaloacetic Acid / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000302:isolation & purification*; D010788:Photosynthesis / Q000502:physiology; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020033:Protein Isoforms; D013696:Temperature
chemical_list: D010940:Plant Proteins; D020033:Protein Isoforms; D062907:Oxaloacetic Acid; D010728:Phosphoenolpyruvate; D010730:Phosphoenolpyruvate Carboxylase