ALLOSTERIC SUBSTRATE INHIBITION IN ARABIDOPSIS MITOCHONDRIAL MALIC ENZYME 1 IS RELEASED BY FUMARATE

MARCOS A TRONCONI; MARIEL GERRARD WHEELER; MARTINATTO, ANDREA; ZUBIMENDI, JUAN PABLO; DRINCOVICH, MARIA F.; CARLOS SANTIAGO ANDREO

Rosario

Conferencia; 8th International Conference for Plant Mitochondrial Biology; 2013

8th International Conference for Plant Mitochondrial Biology

Mitochondrial NAD-dependent malic enzyme (NAD-ME) catalyzes the oxidative decarboxylation of L-malate to yield pyruvate, CO2 and NADH. InArabidopsis, NAD-ME1 and -2 can act as homodimer and heterodimer, and the three enzymatic forms show particular kinetic and regulatory properties.Specifically, NAD-ME1 exhibits a sigmoid L-malate saturation curve and is allosterically activated by fumarate. Because both properties were mappedin a short region on the amino terminal of NAD-ME1 sequence, we hypothesized they are structurally connected. Thus, a set of site-point mutant andchimeric enzymes were generated and characterized by intrinsic fluorescence quenching and kinetic assays. The results strongly support that L-malatebinds to fumarate allosteric site turning to NAD-ME1 in a non-hyperbolic and low-affinity enzyme. Fumarate switches to NAD-ME1 toward ahyperbolic and high-affinity enzyme by competing with the substrate for allosteric site. Thus, fumarate is not a true activator but suppresses theinhibitory effect of L-malate. In addition, the residues Arg50 and Arg84 define the specificity for fumarate or L-malate and are the basis of the homoand heterotrophic effects observed in NAD-ME1. This complex regulation has no been reported for any malic enzyme characterized to date andunderscores the importance of this enzyme as well as C4 organic acids in plant mitochondria metabolism