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...

1. In the absence of sulfate the pH/rate profile for the 4-nitrophenyl acetate hydrolase activity of bovine carbonic anhydrase is complex. The results fit with a microscopic ionization scheme involving two electrostatically interacting groups. The activity depends on the concentration of the basic form of one of these groups. Proton N M R spectra show that the active site residue, His-64, has titration behaviour corresponding to that of the second group of the ionization scheme.2. The addition of increasing concentrations of Na2S04 gradually converts the pH/rate profile to that of a simple titration curve. A pK, of 6.9 is found at 50 mM Na2S04. Concomitantly the titration curve of His-64 changes. The results fit with the microscopic ionization scheme if it is assumed that significant SO:-binding occurs only when both His-64 and the activity-linked group are protonated.3. At constant pH, sulfate behaves as if it inhibits the enzyme only partially. However, data are presented suggesting that the enzyme-sulfate complex is inactive, but the binding of SO:-depends strongly on ionic strength. Thus, above about 25 mM sulfate any further increase of the sulfate concentration is nearly compensated by a decrease of the binding affinity. The binding of a monovalent anion, CI-, also depends on ionic strength, but this dependence is much less prominent than for SO:-.4. Sulfate shows non-competitive behaviour with respect to COz and appears to compete with HCOJ. Iodide and sulfate appear to be mutually competitive. The low pH limit of Ki for I--is 3 mM and about 0.1 mM in the presence of 50 mM Na2SO4 and in the absence of sulfate respectively.5. In the absence of sulfate the Co(I1)-substituted bovine enzyme shows a complex pH/rate profile similar to that of the native enzyme. Changes of the optical spectrum parallel those of the activity. Relaxation rates (I-;') of water protons in the presence of Co(I1) enzyme indicate that the Co(I1) ion might have a H2O ligand at low pH in sulfate-free solutions.6. The effects on sulfate of human carbonic anhydrase I1 (or C) are similar to those on the bovine enzyme.The effects of sulfate on the human 1 (or B) enzyme are smaller.It has been observed already by Roughton and Booth [I] that the catalytic activity of carbonic anhydrase depends both on pH and on the nature and concentrations of the anions present. Extensive subsequent studies on the kinetic and molecular properties of the enzyme have led to proposals of models for the catalytic mechanism incorporating these features [2 -41. The most straightforward hypothesis is the zinc hydroxide model shown in Scheme 1. In this model the pH dependence stems from the ionization of a zinc-bound water molecule. The anion dependence can be described as a competition between OH-and an inhibitory anion for a coordination site on the metal ion. While the evidence for an anion -metal-ion interaction is generally accepted as conclusive, the nature of the activity-linked, ionizing group is still under debate [3,4]. However, it seems certain that this...

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A mechanism model has been presented that can describe most known kinetic properties of carbonic anhydrase isoenzymes I, II, and III. The essential features of this model include: Nucleophilic attack of metal-bound OH- on CO2 to form metal-bound HCO-3. Formation of metal-bound OH- from metal-bound H2O. In isoenzyme II, and probably also in isoenzyme I, this reaction step involves an intramolecular transfer of H+ between the metal site and a titratable histidine residue via a number of hydrogen-bonded H2O molecules. In isoenzyme II, this step limits the maximal rate of catalysis. Also in isoenzyme III, the H2O-splitting step may be rate limiting, but since this isoenzyme has no titratable active-site histidine, H+ transfer may take place directly with components of the solvent. In isoenzymes I and II, rapid H+ transfer between active site and solution proceeds in a reaction between the titratable histidine residue and buffer molecules. The model can also rationalize a variety of observed inhibition patterns.