AN INTERNAL CYSTEINE IS INVOLVED IN THE THIOREDOXIN-DEPENDENT ACTIVATION OF SORGHUM LEAF NADP-MALATE DEHYDROGENASE

The chloroplastic NADP-malate dehydrogenase is activated by thiol/disu lfide interchange with reduced thioredoxins, Previous experiments show ed that four cysteines located in specific N- and carboxyl-terminal ex tensions were implicated in this process, leading to a model where no internal cysteine was involved in activation, In the present study, th e role of the conserved four internal cysteines was investigated, Surp risingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unc hanged activation kinetics, These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cy steines, most probably with one of the two amino-terminal cysteines wh ose mutation similarly accelerates the activation rate, To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino terminal cysteine was mutated in combination with the mutation o f both carboxyl-terminal cysteines were produced and purified, The C29 S/C365A/C377A mutant MDH still needed activation by reduced thioredoxi n, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insen sitive spontaneous activity, leading to the hypothesis that a Cys(24)- Cys(207) disulfide bridge might be formed during the activation proces s, Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are m utated yielded a permanently active enzyme very similar to the previou sly created permanently active C24S/C29S/C365A/C377A mutant. A two-ste p activation model involving a thioredoxin-mediated disulfide isomeriz ation at the amino terminus is proposed.