A 13C Nuclear‐Magnetic‐Resonance Study of CO2‐HCO3− Exchange Catalyzed by Human Carbonic Anhydrase C at Chemical Equilibrium

Abstract

The effects of human carbonic anhydrase C on the ¹³C nuclear magnetic resonance spectra of equilibrium mixtures of ¹³CO₂ and NaH¹³CO₃ were measured at 67.89 MHz. Enzyme-catalyzed CO₂-HCO₃⁻ exchange rates were estimated from the linewidths of the resonances. The results show that: (a) the maximal exchange rates are larger than the maximal turnover rates; (b) the exchange is equally rapid with ¹H₂O or with ²H₂O as solvents; (c) the exchange is equally rapid in the presence or in the absence of added buffers; (d) the apparent substrate binding is weaker than predicted if steady-state K_m values are assumed to represent substrate dissociation constants. The main conclusion concerning the catalytic mechanism of the enzyme is that the proton-transfer processes which limit turnover rates in the steady state are not directly involved in CO₂-HCO₃⁻ exchange. In addition, the results suggest that CO₂-HCO₃⁻ interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc-coordinated OH⁻ with CO₂.