Study of C4-organic acids binding to NAD-malic enzyme 1 from Arabidopsis mitochondria by quenching of protein native fluorescence

MARCOS A. TRONCONI, MARIEL C. GERRARD WHEELER, MARÍA F. DRINCOVICH AND CARLOS S. ANDREO

Salta

Congreso; Latin American Protein Society Meeting; 2010

Sociadad Argentina de Biofísica

Plant mitochondria can use L-malate or fumarate, which accumulate at high levels in plant cells, as respiratory substrates. In part, this plant property is due to the presence of a mitochondrial NAD-dependent malic enzyme (NAD-ME) that displays particular structural and regulatory characteristics. In Arabidopsis, the enzyme acts as two homodimers (NAD-ME1 and NAD-ME2) and also can form a heterodimer1. NAD-ME1 and -2 homodimers are differently regulated by metabolites of central carbon pathways2. In this regard, while the NAD-ME1 activity is positively modulated by fumarate or oxaloacetate, these C4-organic acids not substantially modify the NAD-ME2 activity. In this work, the interaction of NAD-ME1 with fumarate and oxaloacetate was investigated by quenching of tryptophan fluorescence studies. The results indicated that while in NAD-ME2 the quenching process arise from a collisional effect of these metabolites, in NAD-ME1 the fluorescence decay can be explained by a static process that involve the binding of quenchers to the protein surface. The characterization of NAD-ME1 mutant versions revealed that these acids would bind to a single allosteric site on the enzyme. Moreover, the dissociation constants (Kd) values calculated from NAD-ME1 quenching data, indicate that fumarate is the ligand that binds with higher affinity. Finally, the temperature dependence of the thermodynamic parameters (ΔG, ΔH and ΔS) suggests that the fumarate binding process is entropicaly conducted and important alterations in the disposition of lateral chains of aminoacids are produced by the ligand-protein association.