Kinetic and structural studies of Haemophilus influenzae carbonic anhydrase with altered proton transfer capacity

Abstract

The rate‐determining step for CO2 hydration by β‐carbonic anhydrase (β‐CA) is believed to be the loss of a hydrogen ion from the zinc‐bound water to regenerate the zinc‐bound hydroxide ion. Intramolecular acceptors, including a Tyr residue in the active site cleft of Haemophilus influenzae β‐CA (HICA) and a His residue near the exterior of the active site cleft of plant β‐CAs, are hypothesized to facilitate proton transfer from the zinc‐bound water. We report that in HICA the replacement of Tyr83 with Phe decreased kcat ≈30% of wild type, and displays substrate inhibition similar to the analogous Y212F variant of Arabidopsis thaliana β‐CA. Conversely, the replacement of Val87 with His increased the kcat to ≈250% of wild‐type, similar to the kcat of plant β‐CAs. X‐ray crystallographic structures of the V87H variant reveal a unique orientation of Tyr181 to hydrogen bond to the “unique” water molecule in HICA, and alternate “in” and “out” conformations of His87 that could facilitate intermolecular proton transfer to bulk solution; V87H crystals soaked in bicarbonate ion demonstrate that the enzyme still retains the ability to bind bicarbonate in the allosteric site. These results support the hypothesis of proton transfer as the rate‐determining step of β‐CA, and that Tyr and His residues can play important roles in this proton transfer step. This work is supported by NSF grants CHE‐0819686 and MCB‐0741396.