title: Differential fumarate binding to Arabidopsis NAD+-malic enzymes 1 and -2 produces an opposite activity modulation.
abstract: Arabidopsis mitochondria contain two NAD(+)-malic enzymes, NAD-ME1 and NAD-ME2. These proteins have similar affinity for their substrates but display opposite regulation by fumarate, which strongly stimulates NAD-ME1 but inhibits NAD-ME2 activity. Here, the interaction of NAD-ME1 and -2 with fumarate was investigated by kinetic approaches, urea denaturation assays and intrinsic fluorescence quenching, in the absence and presence of NAD(+). Fumarate inhibited NAD-ME2 at saturating, but not at low, levels of NAD(+), and it behaved as competitive inhibitor with respect to L-malate. In contrast, NAD-ME1 fumarate activation was higher at suboptimal NAD(+) concentrations. In the absence of cofactor, the fluorescence of both NAD-ME1 and -2 is quenched by fumarate. However, for NAD-ME2 the quenching arises from a collisional phenomenon, while in NAD-ME1 the fluorescence decay can be explained by a static process that involves fumarate binding to the protein. Furthermore, the residue Arg84 of NAD-ME1 is essential for fumarate binding, as the mutant protein R84A exhibits a collisional quenching by this metabolite. Together, the results indicate that the differential fumarate regulation of Arabidopsis NAD-MEs, which is further modulated by NAD(+) availability, is related to the gaining of an allosteric site for fumarate in NAD-ME1 and an active site-associated inhibition by this C(4)-organic acid in NAD-ME2.
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000, Argentina. tronconi@cefobi-conicet.gov.ar
authors.forename: Marcos A
authors.initials: MA
authors.lastname: Tronconi
authors.forename: Mariel C
authors.initials: MC
authors.lastname: Gerrard Wheeler
authors.forename: María F
authors.initials: MF
authors.lastname: Drincovich
authors.forename: Carlos S
authors.initials: CS
authors.lastname: Andreo
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000187:drug effects / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism / Q000494:pharmacology*; D008291:Malate Dehydrogenase / Q000187:drug effects / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D005650:Fumarates; D009243:NAD; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+)