CHARACTERIZATION OF PEA CHLOROPLASTIC CARBONIC-ANHYDRASE - EXPRESSIONIN ESCHERICHIA-COLI AND SITE-DIRECTED MUTAGENESIS

A cDNA encoding the mature, chloroplast-localized carbonic anhydrase i n pea has been expressed in E. coli. The enzyme is fully active and yi elds of up to 20% of the total soluble protein can be obtained from th e bacteria. This expression system was used to monitor the effects of site-directed mutagenesis of seven residues found within conserved reg ions in the pea carbonic anhydrase amino acid sequence. The effects of these modifications are discussed with respect to the potential of va rious amino acids to act as sites for zinc coordination or intramolecu lar proton shuttles.