The sulfhydryl groups of Cys 269 and Cys 272 are critical for the oligomeric state of chloroplast carbonic anhydrase from Pisum sativum.
Chloroplast carbonic anhydrase is dependent on a reducing environment to retain its catalytic activity. To investigate the properties of the three accessible cysteine residues of pea carbonic anhydrase, mutants were made in which Ala or Ser substituted for C165, C269, and C272. The mutants at position 165 were found to be spectroscopically similarly to the wild-type. They have a high catalytic activity, and are also sensitive to oxidation. In contrast, both C269 and C272 were found to be critical both for the structure and for the catalytic activity. All mutants with substitutions at either of these two positions had to be co-overexpressed with GroES/EL chaperones to give soluble enzyme in Escherichia coli. The k(cat) values were decreased by 2 and 3 orders of magnitude for the C272A and C269A mutants, respectively, and the Km values were increased approximately 7 times. However, the binding of the inhibitor ethoxyzolamide was only slightly weakened. The near-UV CD spectra were found to be changed in both sign and intensity compared to that of the wild-type, and the far-UV spectra indicate some loss of alpha-helix structure. Moreover, the quaternary structure was changed from the wild-type octameric to tetrameric in these mutants. The results indicate that mutation of either of these cysteines causes minor structural changes around at least one of the two tryptophans of the subunit. Furthermore, the data demonstrate that C269 and C272 are involved in the interaction between subunits and are necessary for a proper structure at the tetramer-tetramer interface.
Björkbacka
H
H
Department of Biochemistry, Umeå University, Sweden.
Johansson
I M
IM
Skärfstad
E
E
Forsman
C
C
D002245:Carbon Dioxide / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D003958:Diamide / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005016:Ethoxzolamide / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D018532:Pisum sativum / Q000201:enzymology*; D010720:Phosphines / Q000494:pharmacology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
D002245:Carbon Dioxide; D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases; D003432:Cross-Linking Reagents; D003545:Cysteine; D003958:Diamide; D004228:Dithionitrobenzoic Acid; D005016:Ethoxzolamide; D010720:Phosphines; C080938:tris(2-carboxyethyl)phosphine