Direct evidence for the different roles of the N- and C-terminal regulatory disulfides of sorghum leaf NADP-malate dehydrogenase in its activation by reduced thioredoxin.
Plant NADP-dependent malate dehydrogenase is activated through thiol/disulfide interchange with reduced thioredoxin. Previous studies showed that this process involves the reduction of two different disulfides per subunit: one N-terminal, the other C-terminal. Substitution of regulatory cysteines at each end by site-directed mutagenesis and comparison of activation kinetics of the mutants led us to propose a model for the activation mechanism where the C-terminal end shielded the access to the catalytic residues, whereas the N- terminal end was involved in the slow conformational change of the active site. In the present study, we took advantage of the previous identification of the catalytic histidine residue which can be specifically derivatized by diethyl pyrocarbonate to test the accessibility of the active site. The results clearly show that in the mutants where the C-terminal bridge is open the active site histidine is freely accessible to the reagent, whereas in the mutants where the N-terminal bridge is open, the active site cannot be reached without activation, thus demonstrating the validity of the model.
Issakidis
E
E
Institut de Biotechnologie des Plantes, Université de Paris-Sud, Orsay, France.
Lemaire
M
M
Decottignies
P
P
Jacquot
J P
JP
Miginiac-Maslow
M
M
D004220:Disulfides / Q000378:metabolism*; D004789:Enzyme Activation; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018515:Plant Leaves / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins; D013879:Thioredoxins; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)