Two point mutations convert a catalytically inactive carbonic anhydrase-related protein (CARP) to an active enzyme

FEBS Lett. 1996 Dec 2;398(2-3):322-5. doi: 10.1016/s0014-5793(96)01263-x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetazolamide / pharmacology
  • Amino Acid Sequence
  • Animals
  • Carbon Dioxide / metabolism
  • Carbonic Anhydrase Inhibitors / pharmacology
  • Carbonic Anhydrases / metabolism
  • DNA, Complementary / genetics
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Point Mutation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Zinc / analysis

Substances

  • Carbonic Anhydrase Inhibitors
  • DNA, Complementary
  • Nerve Tissue Proteins
  • Recombinant Proteins
  • Carbon Dioxide
  • Carbonic Anhydrases
  • Zinc
  • Acetazolamide