The catalytic mechanism of carbonic anhydrase

Abstract

Water as a ligand of the zinc in carbonic anhydrase has a pKa of 7 or less and the zinc-bound hydroxide is enhanced as a nucleophile for attack on CO2. The product of catalysis is HCO3− and a proton, and the catalytic pathway as determined for vertebrate isozymes I, II, and III occurs in two separate and distinct stages. The first stage includes the hydration of CO2 and ends with the release of HCO3− from its binding site as a ligand of the zinc; its position is replaced by a water molecule. This process is described by the ratio kcat/Km; for hydration catalyzed by isozyme II, the most efficient of the carbonic anhydrases, kcat/Km is close to diffusion controlled at 108 M−1∙s−1. The second stage is the regeneration of the zinc-bound hydroxide by protolysis of water and release of a proton to the surrounding medium. For carbonic anhydrase II, this proton transfer is rate determining for the maximal turnover number kcat of 106 s−1. Its pathway includes intramolecular proton transfer from the zinc-bound water to His64 in the active-site cavity followed by proton transfer to buffer in solution. For the least efficient of the carbonic anhydrases, isozyme III, kcat/Km near 3 × 105 M−1∙s−1 is not diffusion controlled; nevertheless, proton transfer from zinc-bound water to solution is still rate limiting for a maximal turnover of 104 s−1. Carbonic anhydrase isolated from spinach chloroplasts is quite similar to vertebrate isozyme II in catalytic properties, although it has been found to have almost no sequence homology with the vertebrate carbonic anhydrases. Key words: carbonic anhydrase, CO2, catalytic mechanism, proton transfer, bicarbonate.