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 l3CO2 and NaH13C03 were measured at 67.89 MHz. Enzyme-catalyzed C02-HCO; 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 'H2O or with 2H20 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, values are assumed to represent substrate dissociation constants.The main conclusion concerning the catalytic mechanism of the enzyme is that the protontransfer processes which limit turnover rates in the steady state are not directly involved in C02-HCO: exchange. In addition, the results suggest that COZ-HCO; interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc-coordinated OH-with C02.Carbonic anhydrase is a zinc-containing metalloenzyme catalyzing the reaction C02 + H20 HCO; + H + .( 1) The enzyme is extremely efficient; the turnover number for C 0 2 hydration catalyzed by the human C isoenzyme is lo6 sK1 at 25°C [1,2]. Proton transfer processes are necessarily involved in the reaction. Thus, C02 hydration is associated with the splitting of HzO and the production of H+. The prevalent hypothesis is that rapid H2O splitting is promoted by the metal ion (cf. [3]) which serves as an acceptor for the OH-component of H2O [4]. The H f component of H 2 0 is transferred into the medium perhaps via acceptor groups in the active site [2]. It was pointed out by several authors [l, 5 -81 that a sufficiently rapid release of H' cannot take place unless H' is transported into the medium by some acceptor other than H 2 0 . It was proposed [9-111 that the buffer can have this transport fkction, and this hypothesis has recently gained experimental support [12-141. Thus, at sufficiently low buffer concentration the rate of C02 hydration appears to be limited by a reaction step such as EH + B e E-+ BH'. (2)where B and BH' represent the buffer components [14]. At high buffer concentration reaction 2 is not Abbreviation. NMR, nuclear magnetic resonance. Enzyme. Carbonic anhydrase (EC 4.2.1.1). rate-limiting. However, studies of hydrogen isotope effects on the steady-state kinetic properties of the human C isoenzyme [2] suggested that an intramolecular proton-transfer step separate from the C02-HCO; interconversion limits the rate under these conditions. Schematically, EH' e EH. (3)The presence of such an 'isomerization' step appeared to be supported by product-inhibition patterns [15].To test this hypothetical mechanism further, and to investigate whether proton transfers are involved in the reaction steps associated with substrate-product interconversion, it seemed logical to study the exchange between C02 and HCO; separately. According to our hypothesis this exchange would not involve reactions 2 and 3, but...

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