Identification of an allosteric site for the substrate malate in NAD-malic enzyme 1 from Arabidopsis mitocondria

GERRARD WHEELER, M. C.; ANDREO, C. S.; DRINCOVICH, M. F.; TRONCONI, M. A.

Salta

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Biofisica - CeBEM Workshop on Structural Biology - 3rd Latin American Protein Society Meeting; 2010

Latin American Protein Society

Within Arabidopsis thaliana mitochondria, the L-malate is oxidatively decarboxylated to pyruvate by the NAD-dependent malic enzyme (NAD-ME). This protein is encoded by two nuclear genes, whose products are active as both homo and heterodimers1. Recombinant NAD-ME1 and -2 behaved differently in terms of kinetic reaction mechanism2. In addition, both isoenzymes clearly differed in their regulation by effectors suggesting differential contributions for each one in the organic acid metabolism3. In this work, the interaction of NAD-ME1 with L-malate was investigated by kinetic analysis, fluorescence studies and mutagenesis approach. The results indicated that this substrate exerts a regulatory effect on enzymatic activity, showing a sigmoidal velocity curve. This behavior may be due to the presence of an allosteric site for malate binding in addition to the active site, as indicated by quenching of tryptophan fluorescence experiments. In this regard, the characterization of two NAD-ME1 mutant enzymes allowed to mapp the allosteric site in the primary structure. Interestingly, we identified two arginine residues (R50 and R84) that belong to this regulatory site but contribute differently to the malate binding. These studies are expected to help to elucidate the regulatory network of an enzyme involved in vital processes such as photosynthetic production of carbohydrates, respiration, biosynthesis of proteins and lipids, cellular pH regulation and defense responses. References: 1. Tronconi M., Fahnenstich H., Gerrard Wheeler M., Andreo C., Flügge U., Drincovich M. and Maurino V., Plant Physiol., 146, 1540-1552 (2008) 2. Tronconi M., Gerrard Wheeler M., Maurino V., Drincovich M. and Andreo C., Biochem J., in press 3. Tronconi M., Maurino V., Andreo C. and Drincovich M., J. Biol. Chem., 285, 11870-11879 (2010)