Further evidence for a role of sulfhydryls in the thioredoxin dependent activation of corn NADP-malate dehydrogenase Use of a cysteine free mutant of E. coli thioredoxin

AbstractThe kinetics of activation and reduction of the corn leaf NADP-malate dehydrogenase reveal that plant thioredoxins play a dual role in the activation of this enzyme in the presence of dithiothreitol (e.g. reductive and conformational). A mutant thioredoxin of E. coli in which Cys32 and Cys35 are replaced by an Ala and a Ser residue, respectively, was tested in the activation of corn NADP-malate dehydrogenase either in the presence of DTT or in a light reconstituted system. While native E. coli or plant thioredoxinm strongly activated the NADP-malate dehydrogenase, the mutant thioredoxin was unable to promote any activation of the enzyme. At the same time the mutant thioredoxin stimulated the activity of T7 DNA polymerase. These results clearly confirm that the thioredoxin requirement observed in the activation of NADP-malate dehydrogenase is primarily needed for redox reactions and not for structural/conformational effects as is the case for the T7 DNA polymerase assay