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.
Ruelland
E
E
Institut de Biotechnologie des Plantes, ERS 569 CNRS, Bâtiment 630, Université de Paris-Sud, 91405 Orsay Cedex, France.
Johansson
K
K
Decottignies
P
P
Djukic
N
N
Miginiac-Maslow
M
M
D019943:Amino Acid Substitution; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology; D017931:DNA Primers; D004220:Disulfides / Q000737:chemistry; D002523:Edible Grain / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D050538:Malate Dehydrogenase (NADP+); D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D017931:DNA Primers; D004220:Disulfides; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)