Allosteric substrate inhibition of Arabidopsis NAD-dependent malic enzyme 1 is released by fumarate

MARCOS ARIEL TRONCONI; MARIEL GERRARD WHEELER; ANDREA MARTINATTO; JUAN PABLO ZUBIMENDI; CARLOS SANTIAGO ANDREO; MARIA FABIANA DRINCOVICH

PHYTOCHEMISTRY

PERGAMON-ELSEVIER SCIENCE LTD

Lugar: Amsterdam; Año: 2015 vol. 111 p. 37 - 47

0031-9422

Plant mitochondria can use L-malate and fumarate, which accumulate in large levels, as respiratory substrates. In part, this property is due to the presence of NAD-dependent malic enzymes (NAD-ME) with particular biochemical characteristics. Arabidopsis NAD-ME1 exhibits a non-hyperbolic behavior for the substrate L-malate and its activity is strongly stimulated by fumarate. Here, the possible structural connection between these properties was explored through mutagenesis, kinetics and fluorescence studies. The results indicate that NAD-ME1 presents a regulatory site for L-malate that also can bind fumarate. L-malate binding to this site elicits a sigmoidal and low substrate affinity response. Instead, fumarate binding turns NAD-ME1 in a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was removed or mutated. Hence, fumarate is not really an activator but suppresses the inhibitor effect of L-malate. In addition, the residues Arg50, Arg80 and Arg84 showed different participation in organic acid binding. These residues form a triad which is the basis of the homo and heterotropic effect that characterize to NAD-ME1. The binding of L-malate and fumarate at the same allosteric site is first reported for a malic enzyme and may be related to their role as photosynthetically fixed carbon forms.