2 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 molecul ar mass of 32 950 Da. By introducing two mutations, Arg(117) --> His a nd Glu(115) --> Gln, we created a metal-binding center homologous to t hat in the carbonic anhydrases from the animal kingdom. In contrast to unmodified CARP, this double mutant was isolated as a 1 : 1 zinc-prot ein complex. While unmodified CARP is catalytically inactive, the muta nt catalyzes CO2 hydration with a significantly higher efficiency than the mammalian low-activity carbonic anhydrase isozyme III. The activi ty is strongly inhibited by the powerful and selective carbonic anhydr ase inhibitor, acetazolamide.