Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration of carbon dioxide to bicarbonate and a proton. Catalysis involves an intramolecular proton transfer that delivers an excess proton from the zinc-bound water to an internal proton acceptor, His64. His64 shuttles this proton to the bulk solvent, thus regenerating the active site for the next catalysis. The ability to experimentally increase the rate of proton transfer within the HCA II active site can provide insight into the biophysical properties for this process. The three factors proposed to influence the rate limiting step of proton transfer (k_B) are the active site water network, conformation of His64, and the pK_a of both the zinc bound solvent and His64.

An extensive analysis of the kinetic and structure of wild type and several mutants of HCA II were conducted over a broad pH range. The results show that the enzyme active site is very stable. Several mutants altered the proton shuttle His64 orientation, the water network, and the pK_a of the defined proton donor and acceptor which resulted in altered proton transfer rates. Faster rates of proton transfer were observed in all the mutants. The 2–4-fold increases in proton transfer followed the theoretical values predicated by Marcus theory based on changes in the pK_a. The 7–15 fold increase in proton transfer over wild type showed residue His64 primarily in the inward conformation decreasing the distance to the zinc. The less branched water network with more conventional hydrogen bonds lengths connecting zinc solvent through only two water molecules to His64 appeared in mutants with enhanced proton transfer rates compared to wild type HCA II.

Classically the physiological role of CA is in acid-base balance throughout the body. A recent proposal that CA could catalyze the conversion of nitrite into nitric oxide (NO) a potent vasodilator prompted the design of a system to directly measure NO concentrations in red cell suspensions. The examination of CA alone as well as CA within whole RBC as well as hemoglobin alone did not show sufficient generation of NO at physiological levels of nitrite for vasodilation.