Water exchange at the active site of carbonic anhydrase. A synthesis of the OH- and H2O-models.
We have measured the paramagnetic contribution to the magnetic relaxation rate of solvent protons in highly purified, buffer- and salt-free solutions of Co(2+)-substituted human carbonic anhydrase B (HCAB), as a function of pH in the range 5.5-10 and as a function of magnetic field. We have also measured the optical absorption at 640 nm to characterize the enzyme. The relaxation rates vary with pH much as does the CO(2) hydration activity, increasing with increasing pH. We find that the relaxation rates at all intermediate values of pH can be described as linear combinations of the rates obtained at the extremes of pH used, indicating the existence of low- and high-pH forms of the enzyme with pH-dependent concentrations. The optical data can be similarly represented. The fraction of high-pH form present, determined from either the relaxation or optical data, has a pK(a) of approximately 7.6 when approximated by a single ionization. The data are very similar to that for HCAB in the presence of buffer, in contrast to the bovine enzyme for which the pK(a) is affected substantially by the presence of sulfate. Previous analysis of the high relaxation rates at high pH indicated rapid exchange of Co(2+)-liganded protons, possible only if these exchanging protons were conveyed by water molecules. On the other hand, the present demonstration of the existence of two forms of HCAB in highly purified solutions, coupled with other data, argues strongly for ionization of a water molecule ligand of the metal ion at the active site, with OH(-) as the solvent-donated ligand at high pH. We propose a mechanism of ligand exchange at high pH that reconciles these ostensibly conflicting requirements by invoking a pentacoordinate intermediate having both OH(-) and H(2)O as ligands. Proton exchange can be rapid between these ligands because charge transfer without net ionization can occur, so that the leaving water can carry away the initial OH(-). The low-pH form is a thermal mixture of tetra- and pentacoordinate species, the latter having low relaxation rates by analogy with inhibitor derivatives of the enzyme and model systems. The proposed associative ligand-exchange mechanism reconciles the distinctions between the OH- and H(2)O-models of carbonic anhydrase by merging them, providing the first model is consistent with the observed pH dependence of hydration activity, optical absorption, and solvent magnetic relaxation.
D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D014867:Water / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D014867:Water; D003035:Cobalt; D002256:Carbonic Anhydrases; D015032:Zinc