The autoinhibition of sorghum NADP malate dehydrogenase is mediated by a C-terminal negative charge.

The chloroplastic NADP malate dehydrogenase is completely inactive in its oxidized form and is activated by thiol/disulfide interchange with reduced thioredoxin. To elucidate the molecular mechanism underlying the absence of activity of the oxidized enzyme, we used site-directed mutagenesis to delete or substitute the two most C-terminal residues (C-terminal Val, penultimate Glu, both bearing negative charges). We also combined these mutations with the elimination of one or both of the possible regulatory N-terminal disulfides by mutating the corresponding cysteines. Proteins mutated at the C-terminal residues had no activity in the oxidized form but were partially inhibited when pretreated with the histidine-specific reagent diethyl pyrocarbonate before activation, showing that the active site was partially accessible. Proteins missing both N-terminal regulatory disulfides reached almost full activity without activation upon elimination of the negative charge of the penultimate Glu. These results strongly support a model where the C-terminal extension is docked into the active site through a negatively charged residue, acting as an internal inhibitor. They show also that the reduction of both N-terminal bridges is necessary to release the C-terminal extension from the active site. This is the first report for a thiol-activated enzyme of a regulatory mechanism resembling the well known intrasteric inhibition of protein kinases.