CHARACTERIZATION OF CYSTEINE RESIDUES INVOLVED IN THE REDUCTIVE ACTIVATION AND THE STRUCTURAL STABILITY OF RAPESEED (BRASSICA-NAPUS) CHLOROPLAST FRUCTOSE-1,6-BISPHOSPHATASE

In higher plants, light enhances the activity of chloroplast fructose- 1,6-bisphosphatase via a cascade of thiol/disulfide exchanges. We have examined the structural and functional role of seven conserved cystei ne residues in the rapeseed (Brassica napus) enzyme by site-directed m utagenesis, After lysis of Escherichia coli cells, C53S and C191S vari ants partitioned mainly in the insoluble fraction whereas C96S, C157S, C174S, C179S, and C307S mutants were soluble. Homogeneous preparation s of the latter hydrolyzed fructose 1,6-bisphosphate at similar rates in the presence of 10 mM Mg2+ but only C157S, C174S and C179S mutants were both efficient catalysts at 1 mM Mg2+ and nearly insensitive to d ithiothreitol. These results demonstrate the contribution of Cys53 and Cys191 to the stability of the enzyme and the participation of Cys157 , Cys174 and Cys179 in the reductive process responsive of the light-d ependent regulation. Given that mutations at Cys96 and Cys307 neither destabilize the enzyme nor affect the reductive modulation, their func tion remains unknown. (C) 1997 Academic Press.