Effect of dithiothreitol on the catalytic activity, quaternary structure and sulfonamide-binding properties of an extracellular carbonic anhydrase from Chlamydomonas reinhardtii.
Extracellular carbonic anhydrase from the unicellular green alga Chlamydomonas reinhardtii is an oligomeric protein containing subunits of 36 and 4 kDa which are joined by disulfide bonds to form higher molecular mass oligomers. In this study, the effect of dithiothreitol on some properties of the enzyme were examined. Dithiothreitol caused a 40% activation of the catalytic activity of the enzyme at low concentrations (0.1 mM), but an inactivation of about 85% of the catalytic activity at high (50 mM) concentrations. Chemical cross-linking of the enzyme with dimethyl suberimidate revealed the existence of oligomers containing up to three large subunits and at least two small subunits. Cross-linking analysis of dithiothreitol-treated carbonic anhydrase revealed that 0.1 mM dithiothreitol had no effect on the subunit composition of the enzyme, but 10 or 50 mM caused subunit dissociation, including the apparent complete dissociation of the small subunits from the large subunits. There was a characteristic enhancement of dansylamide fluorescence when this fluorescent sulfonamide bound carbonic anhydrase and the fluorescence enhancement was retained following the dithiothreitol-induced dissociation of the enzyme. These results indicate that disulfide bonds are essential for maintenance of the oligomeric structure of Chlamydomonas reinhardtii carbonic anhydrase, and that the small subunit may be necessary for enhancing catalysis, but not for the binding of sulfonamides to the enzyme.
Husic
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HD
Department of Chemistry, Lafayette College, Easton, PA 18042-1782.
Hsieh
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Berrier
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D002256:Carbonic Anhydrases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D002696:Chlamydomonas / Q000201:enzymology*; D003432:Cross-Linking Reagents; D003619:Dansyl Compounds; D004120:Dimethyl Suberimidate; D004220:Disulfides / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011485:Protein Binding / Q000187:drug effects; D013449:Sulfonamides / Q000378:metabolism
D003432:Cross-Linking Reagents; D003619:Dansyl Compounds; D004220:Disulfides; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D013449:Sulfonamides; C001026:5-dimethylaminonaphthalene-1-sulfonamide; D004120:Dimethyl Suberimidate; D002256:Carbonic Anhydrases; D004229:Dithiothreitol