Interaction of the unique competitive inhibitor imidazole and related compounds with the active site metal of carbonic anhydrase: linkage between pH effects on the inhibitor binding affinity and pH effects on the visible spectra of inhibitor complexes with the cobalt-substituted enzyme.
Previous studies on the interaction of carbonic anhydrase (CA) with the unique CO2 competitive inhibitor imidazole and related compounds were all interpreted as showing that an ionizable water ligand on the metal of this zinc metalloenzyme is not displaced by inhibitor binding. Internal inconsistencies in the pH dependence of binding and the pH dependence of the visible spectra of complexes with cobalt-substituted enzyme prompted us to reinvestigate this binding. Visible spectroscopy was used to measure the binding of imidazole and 1,2,4-triazole to Co(II)-substituted human CA I and active site carboxymethylated human CA I (CmCA I) and the binding of 1,2,4-triazole to bovine CoIICA II. The limiting visible spectra for these enzyme-inhibitor adducts were also computed and examined for pH dependence. It was shown that the pKa of visible spectral changes can be independently predicted from studies on the pH dependence of binding. After consideration of possible contributions from effects of His-200 ionization in CA I and CmCA I, and His-64 in CA II, the pH effects on binding affinity and spectra were found to be of the correct magnitude to establish linkage between binding and an ionization. It was also shown, however, that pH effects on binding and spectra cannot distinguish whether neutral imidazole binds to both ionization forms of the enzyme (Zn-OH2 and Zn-OH) or whether neutral imidazole and its anion both bind to only the acid form of the enzyme, presumably after displacing the water. These findings have implications to the crystallographic interpretations on the imidazole-enzyme complex and to the catalytic mechanism of CO2 hydration.
D000818:Animals; D001665:Binding Sites; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000097:blood*; D002417:Cattle; D003035:Cobalt / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000097:blood; D007700:Kinetics; D013053:Spectrophotometry; D014230:Triazoles / Q000494:pharmacology
D002257:Carbonic Anhydrase Inhibitors; D007093:Imidazoles; D007527:Isoenzymes; D014230:Triazoles; D003035:Cobalt; C029899:imidazole; D002256:Carbonic Anhydrases