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

FEBS Letters

Sjöblom, Björn¹  Wallgren, Katarina¹  Elleby, Björn¹  Jonsson, Bengt-Harald¹  Lindskog, Sven¹ 

[1] Department of Biochemistry, Umeå University, S-90187 Umeå, Sweden

关键词: Carbonic anhydrase-related protein;    Mutagenesis;    CO2 hydration;    Zinc binding;    Acetazolamide;    CA;    carbonic anhydrase;    CARP;    carbonic anhydrase-related protein;    IPTG;    isopropyl thio-β-d-galactoside;   

DOI  :  10.1016/S0014-5793(96)01263-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown