Dual role of ATP and fumarate in the regulation of the major NADP-dependent malic enzyme isoform from Arabidopsis thaliana

CINTIA L. ARIAS; CARLOS S. ANDREO; MARÍA F. DRINCOVICH ; MARIEL C. GERRARD WHEELER

Salta

Congreso; 3rd Latin American Protein Society Meeting; 2010

Latin American Protein Society

A. thaliana contains four malic enzymes (ME 1-4) to catalyze the oxidative decarboxylation of malate in the presence of NADP. These proteins share a high degree of sequence similarity but differ in tissue and cellular compartment expression, kinetic properties and metabolic regulation1-3. ME2 is the only one located in the cell cytosol showing a constitutive pattern of expression1,3 and it is responsible for the major part of NADP-ME activity in mature plant tissues1. In this work, the regulation of this key enzyme by ATP and fumarate was investigated by kinetic analysis, fluorescence studies and mutagenesis approach. ATP acted as an inhibitor of the enzyme activity at high substrates concentration. However, at lower substrates concentration this nucleotide behaves as an activator. Interestingly, the activation peak is reached at malate and NADP concentrations near Km values, which would be important in vivo. In addition, quenching of tryptophan fluorescence experiments indicated that fumarate has two binding sites on ME2. This organic acid produced an increase in ME2 activity by binding to an allosteric site present in this isoform. However, at higher concentrations could also bind to the active site causing a competitive inhibition with respect to the substrate malate. The characterization of ME3 and ME2R115A mutant, which are not activated by fumarate, confirm this hypothesis. Thus, the physiological context might also be controlling the malic enzyme activity in planta, through changes in the metabolite and substrates concentrations.