The Sulfhydryl Groups of Cys 269 and Cys 272 Are Critical for the Oligomeric State of Chloroplast Carbonic Anhydrase from Pisum sativum

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• 作者：Harry Bjö ; rkbacka ; Inga-Maj Johansson ; Eleonore Skä ; rfstad ; and Cecilia Forsman
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：April 8, 1997
• 年：1997
• 卷：36
• 期：14
• 页码：4287 - 4294
• 全文大小：181K
• 年卷期：v.36,no.14(April 8, 1997)
• ISSN：1520-4995

文摘

Chloroplast carbonic anhydrase is dependent on a reducingenvironment to retain its catalyticactivity. To investigate the properties of the three accessiblecysteine residues of pea carbonic anhydrase,mutants were made in which Ala or Ser substituted for C165, C269, andC272. The mutants at position165 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 andC272 were found to be critical both forthe structure and for the catalytic activity. All mutants withsubstitutions at either of these two positionshad to be co-overexpressed with GroES/EL chaperones to give solubleenzyme in Escherichia coli. Thek_cat values were decreased by 2 and 3 orders ofmagnitude for the C272A and C269A mutants, respectively,and the K_m values were increased approximately 7times. However, the binding of the inhibitorethoxyzolamide was only slightly weakened. The near-UV CD spectrawere found to be changed in bothsign and intensity compared to that of the wild-type, and the far-UVspectra indicate some loss of -helixstructure. Moreover, the quaternary structure was changed from thewild-type octameric to tetrameric inthese mutants. The results indicate that mutation of either ofthese cysteines causes minor structuralchanges around at least one of the two tryptophans of the subunit.Furthermore, the data demonstratethat C269 and C272 are involved in the interaction between subunits andare necessary for a properstructure at the tetramer-tetramer interface.