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

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

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

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 (¦¤G, ¦¤H and ¦¤S) 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. References: 1. Tronconi M., Fahnenstich H., Gerrard Wheeler M., Andreo C., Fl¨¹gge U., Drincovich M. and Maurino V., Plant Physiol., 146, 1540-1552 (2008) 2. Tronconi M., Maurino V., Andreo C. and Drincovich M., J. Biol. Chem., 285, 11870-11879 (2010)