Inhibition of the thioredoxin-dependent activation of the NADP-malate dehydrogenase and cofactor specificity

The chloroplastic NADP-malate dehydrogenase is activated by reduction of it s N- and C-terminal disulfides by reduced thioredoxin. The activation is in hibited by NADP(+), the oxidized form of the cofactor. Previous studies sug gested that the C-terminal disulfide was involved in this process. Recent s tructural data pointed toward a possible direct interaction between the C t erminus of the oxidized enzyme and the cofactor. In the present study, the relationship between the cofactor specificity for catalysis and for inhibit ion of activation has been investigated by changing the cofactor specificit y of the enzyme by substitution of selected residues of the cofactor-bindin g site. An NAD-specific thiol-regulated MDH was engineered. Its activation was inhibited by NAD(+) but no longer by NADP(+). These results demonstrate that the oxidized cofactor is bound at the same site as the reduced cofact or and support the idea of a direct interaction between the negatively char ged C-terminal end of the enzyme and the positively charged nicotinamide ri ng of the cofactor, in agreement with the structural data. The structural r equirements for cofactor specificity are modeled and discussed.