title: Two point mutations convert a catalytically inactive carbonic anhydrase-related protein (CARP) to an active enzyme.
abstract: A murine carbonic anhydrase-related protein (CARP) has been expressed in Escherichia coli and purified to near homogeneity. The polypeptide chain consists of 290 amino acid residues and has a calculated molecular mass of 32,950 Da. By introducing two mutations, Arg117 --> His and Glu115 --> Gln, we created a metal-binding center homologous to that in the carbonic anhydrases from the animal kingdom. In contrast to unmodified CARP, this double mutant was isolated as a 1:1 zinc-protein complex. While unmodified CARP is catalytically inactive, the mutant catalyzes CO2 hydration with a significantly higher efficiency than the mammalian low-activity carbonic anhydrase isozyme III. The activity is strongly inhibited by the powerful and selective carbonic anhydrase inhibitor, acetazolamide.
authors.affiliation: Department of Biochemistry, Umeå University, Sweden.
authors.forename: B
authors.initials: B
authors.lastname: Sjöblom
authors.forename: B
authors.initials: B
authors.lastname: Elleby
authors.forename: K
authors.initials: K
authors.lastname: Wallgren
authors.forename: B H
authors.initials: BH
authors.lastname: Jonsson
authors.forename: S
authors.initials: S
authors.lastname: Lindskog
mesh_terms: D000086:Acetazolamide / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000032:analysis
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases; D015032:Zinc; D000086:Acetazolamide