STRUCTURAL AND KINETIC CHARACTERIZATION OF A PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC) FROM CITRUS FRUIT

FALCONE FERREYRA, M. LORENA; PEROTTI, VALERIA; IGLESIAS, ALBERTO A; PODESTÁ, FLORENCIO E

Pinamar, Buenos Aires

Congreso; · XLI Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2005

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

The increase in PEPC activity and protein level in frost-exposed Valencia orange fruit (Citrus sinensis L. Osbeck) has been reported before. In this work, the kinetic and structural properties of PEPC were analyzed in both control and frost-damaged fruit. In common with most other plant PEPCs, the enzyme showed lower Km(PEP) at pH 8 than pH 7, being these values approximately 50% lower in frozen fruits respect to controls. The enzyme was modulated by several metabolites and the effects were generally more pronounced at pH 7 than 8. At pH 7, control fruit PEPC was activated by glucose-6-phosphate and potently inhibited by malate (with a low I50 of 8 mM), aspartate, glutamate and succinate. In contrast, frost-damaged fruit PEPC showed much lower sensitivity to these effectors with a pronounced increase in I50 for malate (125-fold). Citrus fruit PEPC appears to be an oligomeric complex with a mass estimated in 500-600 kDa, composed of 110-kDa subunits and unusual 74-Da polypeptides that were observed both in immunoblots of denaturing crude extracts and silver stained SDS-pages of PEPC activity bands excised from non-denaturing gels. These results suggest that citrus fruit PEPC is different from common plant PEPCs in its structural properties, while the different sensitivity to malate could indicate changes in the phosphorylation state in control and frost-damaged fruit PEPC.