Molecular basis of ionic strength effects: interaction of enzyme and sulfate ion in CO2 hydration and HCO3- dehydration reactions catalyzed by carbonic anhydrase II.
CO2 hydration and HCO3- dehydration reactions catalyzed by carbonic anhydrase II have been examined at various concentrations of sodium sulfate with a stopped-flow technique. We find that at low ionic strength CO2 hydration and HCO3- dehydration reaction rates remain unaffected by varying the salt concentration at pH higher than 7.0, while the reaction rates decrease with increasing ionic strength at low pH. For CO2 hydration reactions, salt effects reside only in the kcat term, not in the Km term, whereas for HCO3- dehydration reactions, salt effects reside only in the Km term, not in the kcat term. In this regime, the salt concentration dependence of the turnover rate for CO2 hydration at low pH is attributed to an electrostatic effect on the ionization constants of the enzyme and/or enzyme-substrate complex, which in turn affect the pH profile of kcat. The rates of the bimolecular interaction between the uncharged CO2 molecule and carbonic anhydrase II at high pH are unaffected by low salt concentration while the rates of the bimolecular interaction of HCO3- with enzyme at low pH decrease with increasing salt concentration, consistent with a negative salt effect on an electrostatically enhanced diffusion of the negatively charged substrate to the positively charged active site. These bimolecular reactions between enzyme and substrate at low ionic strength obey rate equations derived from the Debye-Hückel limiting law and the transition-state theory. Simple linear relationships between the logarithm of the catalytic constant, log kdenz, and the square root of the ionic strength were established.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000097:blood*; D002417:Cattle; D004912:Erythrocytes / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D013431:Sulfates / Q000494:pharmacology*
D001639:Bicarbonates; D013431:Sulfates; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases