Sites of interaction of thioredoxin with sorghum NADP-malate dehydrogenase

The activation pathway of the chloroplastic NADP-dependent malate dehydroge nase (MDH) by reduced thioredoxin has been examined using a method based on the mechanism of thiol/disulfide interchanges, i.e. the transient formatio n of a mixed disulfide between the target and the reductant. This disulfide can be stabilized when each of the partners is mutated in the less reactiv e cysteine of the disulfide/dithiol pair. As NADP-MDH has two regulatory di sulfides per monomer, four different single cysteine mutants were examined, two for the C-terminal bridge and two for the N-terminal bridge. The resul ts clearly show that the nucleophilic attack of thioredoxin on the C-termin al bridge proceeds through the formation of a disulfide with the most exter nal Cys377. The results are less clear-cut for the N-terminal cysteines and suggest that the Cys24-Cys207 disulfide bridge previously proposed to be a n intermediary step in MDH activation can form only when the C-terminal dis ulfide is reduced. (C) 2001 Federation of European Biochemical Societies. P ublished by Elsevier Science B.V. All rights reserved.