Kinetics, anion binding and mechanism of Co(II)-substituted bovine muscle carbonic anhydrase.
The binding of N3- to Co(II)-substituted bovine carbonic anhydrase III was measured at various pH values by spectrophotometric titrations. The apparent Ki values were found to increase with pH in the studied range between pH 5.8 and 8.9. The inhibition of CO2 hydration by N-3 was found to be essentially uncompetitive at all investigated pH values (pH 6.3-8.9). The Ki values for the inhibition of kcat are much smaller than those obtained in the spectrophotometric titrations indicating that an enzyme form with a high affinity for N-3, presumably having a metal-bound H2O, accumulates in the steady state at saturating CO2 concentrations. Assuming that the low pH limit of Ki = 9 microM for the inhibition of kcat represents the affinity of N-3 for the Co(II)-OH2 form, a pKa value near 5 can be estimated for Co(II)-bound water from the pH dependence of N-3 binding in the absence of CO2. Measurements of time-resolved absorption spectra during CO2 hydration in the presence of a low N-3 concentration showed the transient appearance of the characteristic spectrum of the enzyme-N-3 adduct clearly demonstrating the accumulation in the steady state of an enzyme form with a high affinity for N-3. In similar experiments without inhibitor the transient formation of a spectral form corresponding to a Co(II)-OH2 species has been demonstrated. This spectral form is rather featureless lacking the absorption maxima at 618 nm and 640 nm characteristic of the Co(II)-OH- species. Our results strongly support the hypothesis that the rate-limiting step in CO2 hydration catalyzed by carbonic anhydrase III is the protolysis of metal-bound water.
D000818:Animals; D000838:Anions; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002417:Cattle; D003035:Cobalt; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009573:Nitrites / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry
D000838:Anions; D007527:Isoenzymes; D009573:Nitrites; D002245:Carbon Dioxide; D003035:Cobalt; D002256:Carbonic Anhydrases