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

MARTIN, MARIANA; RIUS, SEBASTIÁN P.; PODESTÁ, F. E.

PLANT PHYSIOLOGY AND BIOCHEMISTRY

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

Año: 2011 vol. 49 p. 646 - 653

0981-9428

Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found tocoexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa formwas not the result of proteolytic cleavage of the larger form since extraction methods reported to preventPEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leafextracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically andkinetically characterized and showed parameters compatible with a fully active enzyme. The specificactivity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation ofphosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plantPEPCKs. 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 bysuccinate. A break in the Arrhenius plot at about 30
C indicates that PEPCK structure is responsive tochanges in temperature. The results indicate that pineapple leaves contain two PEPCK forms. Thebiochemical characterization of the smaller isoform performed in this work suggests that it couldparticipate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.