CEFOBI   05405
CENTRO DE ESTUDIOS FOTOSINTETICOS Y BIOQUIMICOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Identification of an allosteric site for the substrate malate in NAD-malic enzyme 1 from Arabidopsis mitochondria
Autor/es:
MARIEL C. GERRARD WHEELER, CARLOS S. ANDREO, MARÍA F. DRINCOVICH AND MARCOS A. TRONCONI.
Lugar:
Salta
Reunión:
Congreso; Latin American Protein Society Meeting; 2010
Institución organizadora:
Sociadad Argentina de Biofísica
Resumen:
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.