Allosteric substrate inhibition in Arabidopsis mitochondrial malic enzyme 1 is released by fumarate

TRONCONI, M. A.; GERRARD WHEELER, M. C.; MARTINATTO, A.; ZUBIMENDI, J. P.; DRINCOVICH, M. F.; ANDREO, C. S.

Rosario

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

Mitochondrial NAD-dependent malic enzyme (NAD-ME) catalyzes the oxidative decarboxylation of L-malate to yield pyruvate, CO2 and NADH. In Arabidopsis, 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 mapped in a short region on the amino terminal of NAD-ME1 sequence, we hypothesized they are structurally connected. Thus, a set of site-point mutant and chimeric enzymes were generated and characterized by intrinsic fluorescence quenching and kinetic assays. The results strongly support that L-malate binds to fumarate allosteric site turning to NAD-ME1 in a non-hyperbolic and low-affinity enzyme. Fumarate switches to NAD-ME1 toward a hyperbolic and high-affinity enzyme by competing with the substrate for allosteric site. Thus, fumarate is not a true activator but suppresses the inhibitory effect of L-malate. In addition, the residues Arg50, Arg80 and Arg84 form a triad that defines the specificity for fumarate or L-malate and is the basis of the homo and heterotrophic effects observed in NAD-ME1. This complex regulation has not been reported for any malic enzyme characterized to date and underscores the importance of this enzyme as well as C4 organic acids in plant mitochondria metabolism.