CYSTEINE-153 IS REQUIRED FOR REDOX REGULATION OF PEA CHLOROPLAST FRUCTOSE-1,6-BISPHOSPHATASE

Chloroplastic fructose-1,6-bisphosphatases are redox regulatory enzyme s which are activated by the ferredoxin thioredoxin system via the red uction/isomerization of a critical disulfide bridge, All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 am ino acids, In order to gain more information on the nature of the regu latory site, five cysteine residues (Cys(49), Cys(153), Cys(173), Cys( 178) and Cys(190)) have been modified individually into serine residue s by site-directed mutagenesis, While mutations C173S and C178S strong ly affected the redox regulatory properties of the enzyme, the most st riking effect was observed with the C153S mutant which became permanen tly active and redox independent, On the other hand, the C190S mutant retained most of the properties of the wild-type enzyme (except that i t could now also be partially activated by the NADPH/NTR/thioredoxin h system), Finally, the C49S mutant is essentially identical to the wil d-type enzyme, These results are discussed in the light of recent crys tallographic data obtained on spinach FBPase [Villeret et al, (1995) B iochemistry 34, 4299-4306].