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

MARCOS TRONCONI; MARIEL GERRARD WHEELER; ANDREA MARTINATTO; JUAN PABLO ZUBIMENDI; ANDREO, CS; 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 can also bind fumarate. L-malate binding to this site elicits a sigmoidal and low substrate-affinity response. Instead, fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was either removed or altered. Hence, fumarate is not really an activator but suppresses the inhibitory effect of L-malate. In addition, residues Arg50, Arg80 and Arg84 showed different participationroless in organic acid binding. These residues form a triad, which is the basis of the homo and heterotrophic effects that characterize NAD-ME1. The binding of L-malate and fumarate at the same allosteric site is here reported for the first time for a malic enzyme and clearly indicates an important role for of NAD-ME1 in the processes that control the flow of C4 organic acids in Arabidopsis mitochondrial metabolism