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

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.