Metalloenzymes and Model Systems. Carbonic Anhydrase: Solvent and Buffer Participation, Isotope Effects, Activation Parameters and Anionic Inhibition

Abstract

The interconversion between CO2 and HCO 3 - in the presence of bovine carbonic anhydrase (BCA) was studied by initial rate measurements using a stopped-flow indicator method. The results analyzed in terms of the Michaelis-Menten Scheme exhibited a sigmoidal variation of the turnover number (kcat) against pH with a pKa value of about 6.8 at 25.0°. For CO2 hydration large kcat values were observed in the high pH region, while for HCO 3 - dehydration large kcat values were observed in the low pH region. For both substrates Km values did not show any significant variation with pH in the region studied. Solvent deuterium isotope effects were found to be between 2.5 and 3.0 at 25.0° for both kcat and Km, and did not vary significantly with change of pH or substrate. Activation parameters for CO2 hydration catalyzed by H2O, OH-, and BCA were obtained in the temperature range 7.0°–35.0°C. For BCA catalysis, temperature effects were confined largely to kcat. Inhibition of BCA by monoanions was studied over a pH range of 6.6 to 9.0 for CO2 hydration, and 6.6 to 7.0 for HCO 3 - dehydration. Anions were found to exhibit linear mixed inhibition of CO2 hydration at low pH, linear uncompetitive inhibition of CO2 hydration at high pH, and linear competitive inhibition of HCO 3 - dehydration at all pH values studied. The implications of these results are discussed, a formal kinetic scheme is proposed, and a mechanism presented to account for these observations.