The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP -MDH) in the activation of the enzyme has been investigated through the exa mination of the ability of this residue to form mixed disulphides with thio redoxin mutated at either of its two active-site cysteines. The R-type Chla mydomonas thioredoxin was used, because it has no additional cysteines in t he primary sequence besides the active-site cysteines. Both thioredoxin mut ants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of it s N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experi ments confirm that the opening of the N-terminal disulphide loosens the int eraction between subunits, making Cys-207, located at the dimer contact are a, more accessible. (C) 1999 Federation of European Biochemical Societies.