| Carbonic anhydrases (CA) are ubiquitously expressed metalloenzymes that are found in all organisms, ranging from bacteria to humans. Human CA II (HCA II) is the most well-studied and utilizes a zinc-hydroxide mechanism to catalyze the reversible hydration of carbon dioxide to produce bicarbonate and a proton. Catalysis involves an intramolecular proton transfer event that delivers an excess proton from the zinc-bound water to an internal proton acceptor, His64. His64 then shuttles this proton to the bulk solvent, thus regenerating the active site for the next round of catalysis.;An extensive analysis of the structural and kinetic stability of wild type and several mutants of HCA II was conducted over a broad pH range. The results show that the enzyme, and the water network in the active site, is extremely stable. It is also the first observation of sulfate ion binding in the active site of wild type HCA II. Attempts to disrupt not only the proton shuttle His64, but other residues involved in stabilizing the water network were also successful as reflected in changes of the measured proton transfer rates. Overall, the results give insights into the structural requirements for efficient proton transfer as catalyzed by CA. To directly observe the active site waters and protonation state of His64, perdeuterated wild type HCA II was produced, crystallized and the X-ray structure determined. This work lays the foundation for future proposed neutron diffraction experiments.;Classical, clinically used CA inhibitors (CAI) are not very water-soluble and this feature has implications for bioavailability of these drugs. The X-ray structures of two novel, water-soluble CAIs bound to HCA II were determined. They reveal that incorporation of spacer groups and fluorines can change the binding modes of CAIs. This work has implications for the clinical use and bioavailability of systemically applied CAIs and for targeting different isozymes of HCA.;A CA from mosquito larvae (AaCA1) was also expressed, purified, and structurally and kinetically characterized. AaCA1 is a high activity CA that shows inhibition with all the classical sulfonamide-based CAIs. This enzyme represents an interesting new drug target for the control of mosquito populations and further understanding of CA function in other organisms. |
