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 o xidized form and is activated by thiol/disulfide interchange with reduced t hioredoxin, To elucidate the molecular mechanism underlying the absence of activity of the oxidized enzyme, we used site-directed mutagenesis to delet e or substitute the two most C-terminal residues (C-terminal Val, penultima te Glu, both bearing negative charges). We also combined these mutations wi th the elimination of one or both of the possible regulatory N-terminal dis ulfides by mutating the corresponding cysteines. Proteins mutated at the C- terminal residues had no activity in the oxidized form but were partially i nhibited when pretreated with the histidine-specific reagent diethyl pyroca rbonate before activation, showing that the active site was partially acces sible. Proteins missing both N-terminal regulatory disulfides reached almos t 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 char ged residue, acting as an internal inhibitor, They show also that the reduc tion of both N-terminal bridges is necessary to release the C-terminal exte nsion from the active site. This is the first report for a thiol-activated enzyme of a regulatory mechanism resembling the well known intrasteric inhi bition of protein kinases.