Inhibition of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle

Using stopped flow methods, we have measured the steady state rate constants and the inhibition by N3- and I- of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle. Also, using fluorescence quenching of the enzyme at 330 nm, we have measured the binding of the sulfonamide chlorzolamide to cat carbonic anhydrase III. Inhibition by the anions was uncompetitive at pH 6.0 and was mixed at higher values of pH. The inhibition constant of azide was independent of pH between 6.0 and 7.5 with a value of KIintercept = 2 X 10(-5) M; the binding constant of chlorzolamide to cat carbonic anhydrase III was also independent of pH in the range of 6.0 to 7.5 with a value Kdiss = 2 X 10(-6) M. Both of these values increased as pH increased above 8. There was a competition between chlorzolamide and the anions N-3 and OCN- for binding sites on cat carbonic anhydrase III. The pH profiles for the kinetic constants and the uncompetitive inhibition at pH 6.0 can be explained by an activity-controlling group in cat carbonic anhydrase III with a pKa less than 6. Moreover, the data suggest that like isozyme II, cat isozyme III is limited in rate by a step occurring outside the actual interconversion of CO2 and HCO3- and involving a change in bonding to hydrogen exchangeable with solvent water.     

Buffer dependence of CO2 hydration catalyzed by human carbonic anhydrase I.

The steady-state kinetics of CO2 hydration catalyzed by human carbonic anhydrase I (carbonate hydro-lyase, EC 4.2.1.1) has been investigated at three pH values corresponding to different parts of the pH-rate profile. Two buffer systems with similar pKa values were used at each pH. The results show that the catalyzed rates depend on the buffer concentration but also on the chemical nature of the buffer. For example, at pH 8.8 the buffer 1,2-dimethylimidazole behaves formally as a second substrate in a 'ping-pong' mechanism yielding a maximal kcat value of 2.2 x 10(5) s-1, whereas much lower rates were obtained with Taps buffers. Similarly, at pH 7.3 1-methylimidazole yields higher rates than Mops and at pH 6.3 3,5-lutidine is more efficient than Mes. Non-Michaelis-Menten kinetics were observed with all buffers except 1,2-dimethylimidazole. In addition, while the apparent buffer activation by 1,2-dimethylimidazole can be described by a single Km value of 26 mM, the Mes concentration dependence is consistent with the presence of two components of similar magnitudes with Km values of 45 mM and 0.15 mM. These results are interpreted within the framework of the 'zinc-hydroxide' mechanism in terms of multiple pathways for the rate-contributing transfer of a proton from the zinc-bound water molecule, formed during CO2/HCO3- interconversion, to the reaction medium, thus, regenerating zinc-bound OH-.     

Molecular basis of the oxygen exchange from CO2 catalyzed by carbonic anhydrase III from bovine skeletal muscle

The exchange of 18O from CO2 to H2O in aqueous solution is caused by the hydration-dehydration cycle and is catalyzed by the carbonic anhydrases. In our previous studies of 18O exchange at chemical equilibrium catalyzed by isozymes I and II of carbonic anhydrase, we observed simple first-order depletion of 18O from CO2 with the 18O distribution among the species C18O18O, C16O18O, and C16O16O described by the binomial expansion (i.e., a random distribution of 18O). Using membrane-inlet mass spectrometry, we have measured 18O exchange between CO2 and H2O catalyzed by native zinc-containing and cobalt(II)-substituted carbonic anhydrase III from bovine skeletal muscle near pH 7.5. The distributions of 18O in CO2 deviate from the binomial expansion and are accompanied by biphasic 18O-exchange patterns; moreover, we observed regions in which 18O loss from CO2 was faster than 18O loss from HCO3-. These data are interpreted in terms of a model that includes 18O loss from an enzyme-substrate or intermediate complex. We conclude that more than one 18O can be lost from CO2 per encounter with the active site of isozyme III, a process that requires scrambling of oxygens in a bicarbonate-enzyme complex and cycling between intermediate complexes. This suggests that the rate of dissociation of H2(18)O (or 18OH-) from isozyme III is comparable to or faster than substrate and product dissociation.     

Hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle

We report three experiments which show that the hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle occurs at a site on the enzyme different than the active site for CO2 hydration. This is in contrast with isozymes I and II of carbonic anhydrase for which the sites of 4-nitrophenyl acetate hydrolysis and CO2 hydration are the same. The pH profile of kcat/Km for hydrolysis of 4-nitrophenyl acetate was roughly described by the ionization of a group with pKa 6.5, whereas kcat/Km for CO2 hydration catalyzed by isozyme III was independent of pH in the range of pH 6.0-8.5. The apoenzyme of carbonic anhydrase III, which is inactive in the catalytic hydration of CO2, was found to be as active in the hydrolysis of 4-nitrophenyl acetate as native isozyme III. Concentrations of N-3 and OCN- and the sulfonamides methazolamide and chlorzolamide which inhibited CO2 hydration did not affect catalytic hydrolysis of 4-nitrophenyl acetate by carbonic anhydrase III.     

Inhibition by cupric ions of the hydration of CO2 catalyzed by carbonic anhydrase II

The inhibition by cupric ions of the hydration of CO₂ catalyzed by carbonic anhydrase II is interesting because of the results of Tuet al. obtained at chemical equilibrium, indicating that Cu²⁺ inhibits specifically a proton transfer in the catalytic pathway. We have measured this inhibition at steady state, using stopped-flow methods. The inhibition by Cu²⁺ of the hydration of CO₂ catalyzed by carbonic anhydrase II had aK _I near 1×10^?6 M atpH 7.0 and gave inhibition that is noncompetitive atpH 6.0 and mixed, but close to uncompetitive, atpH 6.8. ThepH dependence of this binding is consistent with a binding site for Cu²⁺ on the enzyme with apK _a near 7. The binding interaction between Cu²⁺ and the fluorescent inhibitor 5-dimethylaminonaphthalene-l-sulfonamide on carbonic anhydrase II was noncompetitive, indicating that the binding site for Cu²⁺ is distinct from the coordination sphere of zinc in which the actual interconversion of CO₂ and HCO ₃ ^? and the binding of sulfonamides takes place.     

Characterization of human carbonic anhydrase III from skeletal muscle

A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.     

Activation parameters for the carbonic anhydrase II-catalyzed hydration of CO2

We have determined the activation parameters of kcat and kcat/Km for the carbonic anhydrase II-catalyzed hydration of CO2. The enthalpy and entropy of activation for kcat is 7860 +/- 120 cal mol-1 and -3.99 +/- 0.42 cal mol-1 K-1, respectively, for the human enzyme. Results for the bovine enzyme were statistically indistinguishable from those of the human enzyme. The entropy of activation of kcat for the human enzyme was further decomposed into partially compensating electrostatic(es) (delta S*es = +15.1 cal mol-1 K-1) and nonelectrostatic(nes) (delta S*nes = -19.1 cal mol-1 K-1) terms. Computer simulations of a formal kinetic mechanism for carbonic anhydrase II-catalyzed CO2 hydration show that 82% of the temperature effect on kcat can be attributed to the temperature effect on the intramolecular proton transfer step. The reported activation parameters are consistent with a substantial enzyme or active site solvent conformational change in the transition state of the intramolecular proton transfer step, and is consistent with the mechanism of proton transfer proposed by Venkatasubban and Silverman (Venkatasubban, K. S., and Silverman, D. N. (1980) Biochemistry 19, 4984-4989).     

Immunoelectron microscopic localization of carbonic anhydrase III in rat skeletal muscle

Summary The subcellular distribution of carbonic anhydrase III in rat soleus and vastus lateralis muscles was studied using an immunogold technique. The enzyme protein was found to be distributed diffusely in the cytoplasm of skeletal muscle cells. Red skeletal muscle (mainly type I fibers) revealed very strong immunogold staining whereas in white muscle (mainly type II fibers) gold particles were almost completely absent. No immunoreaction was observed in mitochondria or in other intracellular organelles.     

A 13C nuclear-magnetic-resonance study of CO2-HCO3-exchange catalyzed by human carbonic anhydrase C at chemical equilibrium.

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-HCO-3 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 Km 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-HCO-3 exchange. In addition, the results suggest that CO2-HCO-3 interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc-coordinated OH- with CO2.     

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.     

Inhibition by cupric ions of the hydration of CO2 catalyzed by carbonic anhydrase II

The inhibition by cupric ions of the hydration of CO₂ catalyzed by carbonic anhydrase II is interesting because of the results of Tuet al. obtained at chemical equilibrium, indicating that Cu²⁺ inhibits specifically a proton transfer in the catalytic pathway. We have measured this inhibition at steady state, using stopped-flow methods. The inhibition by Cu²⁺ of the hydration of CO₂ catalyzed by carbonic anhydrase II had aK _I near 1×10^–6 M atpH 7.0 and gave inhibition that is noncompetitive atpH 6.0 and mixed, but close to uncompetitive, atpH 6.8. ThepH dependence of this binding is consistent with a binding site for Cu²⁺ on the enzyme with apK _a near 7. The binding interaction between Cu²⁺ and the fluorescent inhibitor 5-dimethylaminonaphthalene-l-sulfonamide on carbonic anhydrase II was noncompetitive, indicating that the binding site for Cu²⁺ is distinct from the coordination sphere of zinc in which the actual interconversion of CO₂ and HCO ₃ ^– and the binding of sulfonamides takes place.     

Exchange of labeled nuclei in the CO2--HCO3--solvent system catalyzed by carbonic anhydrase.

Silverman et al. (1979. J. Am. Chem. Soc. 101:6734-6740) have reported measurements of the loss of 18O to solvent from the isotopically labeled CO2--HCO3-system and of the mixing of 18O and 13C labels within the system, as catalyzed by human carbonic anhydrase C in the pH range 6-8. This work is an extension of earlier work (Silverman and Tu. 1976. J. Am. Chem. Soc. 98:978-984) on the very similar bovine enzyme. The more recent work is analyzed by its authors in terms of the "hydroxide" model for the apparent pH-dependence of enzymatic activity, a model in which the pH-dependence is associated with the presumed ionization of an H2O ligand of the active-site metal ion to OH-. From a comparison of their data with a solution of the coupled differential equations that describe the kinetics of isotope exchange in terms of the model, Silverman et al. derived a pH-dependent rate of exchange for the water molecule which is formed at the active site of the enzyme during dehydration. By contrast, using the same data and a model in which active enzyme has a water molecule on the metal ion at the active site, and similar differential equations, we derive a value for the rate of exchange of water that is pH-independent. This model has the attraction that it explains the magnetic relaxation rate of solvent water protons in the Co2+-substituted enzyme, whereas the hydroxide mechanism cannot explain these data without the introduction of unfounded ad hoc assumptions; further, the presence of an OH- ligand of the metal has never been demonstrated. We also include an analysis of analogous data for the bovine enzyme. One result of our analysis is that the pKa for activity of the enzyme samples used is near 6.0, implying that the bulk of the data were taken when the enzyme was essentially all active. It is straightforward to account for the pH-dependence of the data near and below the pKa by using an empirically-derived value for the pKa. However, we have recently developed a model for the low pH (inactive) enzyme that has been successful in interpreting a wide range of data, and we show that this new view can explain the few points at low pH quite adequately. Additionally, we consider the recent kinetic results for the human C enzyme, obtained at chemical equilibrium by studies of the linewidths of nuclear magnetic resonances of 13C in labeled substrate (Simonsson et al. 1979. Eur. J. Biochem. 93:409-417) and show that these experiments and those of Silverman et al. are all consistent with kinetic data from nonequilibrium stopped-flow experiments, viewed in terms of our model, in the limit of low substrate concentration. Results at higher concentrations indicate that the Michaelis constants and equilibrium constants differ somewhat.     

Mechanism of cyanamide hydration catalyzed by carbonic anhydrase II suggested by cryogenic X-ray diffraction

The three-dimensional structure of a possible intermediate in the hydration reaction of cyanamide to urea catalyzed by human carbonic anhydrase II (hCAII) has been determined by cryocrystallographic techniques. The crystal structure shows that two different adducts are formed under the experimental conditions and that they have different occupancy in the crystal. The high occupancy form consists of a binary hCAII-cyanamide complex where the substrate has replaced the zinc-bound hydroxide anion present in the native enzyme, maintaining the tetrahedral geometry around the metal ion. The second, low-occupancy form consists of a hCAII-cyanamide-water ternary complex where the catalytic zinc ion, still being bound to cyanamide, is approached by a water molecule in a five-coordinate adduct. While the first form can be considered a nonproductive complex, the second form may represent an intermediate state of the catalyzed reaction where the water molecule is about to perform a nucleophilic attack on the zinc-activated cyanamide substrate. The structural evidence is consistent with the kinetic data previously reported about this recently described hydrolytic reaction catalyzed by hCAII, and indicates that a different mechanism with respect to that generally accepted for the physiologic carbon dioxide hydration reaction may be adopted by the enzyme, depending on the substrate chemical properties.     

A comparison of the mechanisms of CO2 hydration by native and Co2+-substituted carbonic anhydrase II

We have measured the pH dependence of kcat and kcat/Km for CO2 hydration catalyzed by both native Zn2+-and metallo-substituted Co2+-bovine carbonic anhydrase II in the absence of inhibitory ions. For the Zn2+-enzyme, the pKa values controlling kcat and kcat/Km profiles are similar, but for the Co2+-enzyme the values are about 0.6 pH units apart. Computer simulations of a metal-hydroxide mechanism of carbonic anhydrase suggest that the data for both native and Co2+-carbonic anhydrase can be accounted for by the same mechanism of action, if we postulate that the substitution of Co2+ for Zn2+ in the active site causes a separation of about 0.6 pH units in the pKa values of His-64 and the metal-bound water molecule. We have also measured the activation parameters for kcat and kcat/Km for Co2+-substituted carbonic anhydrase II-catalyzed CO2 hydration and have compared these values to those obtained previously for the native Zn2+-enzyme. For kcat and kcat/Km we obtain an enthalpy of activation of 4.4 +/- 0.6 and approximately 0 kcal mol-1, respectively. The corresponding entropies of activation are -18 +/- 2 and -27 +/- 2 cal mol-1 K-1.     

Regulation of carbonic anhydrase III by thyroid hormone: opposite modulation in slow- and fast-twitch skeletal muscle

This laboratory previously reported that a major 30 kilodalton (kDa) protein of the soluble cytoplasmic fraction of the rat slow-twitch soleus muscle is modulated by thyroid hormone. This protein has been purified and a portion of the primary structure has been determined. The sequence analysis suggested that the 30-kDa protein is carbonic anhydrase III (CA III; EC 4.2.1.1). The reaction of the protein with a CA III specific antibody and the similar modulation of CA III by thyroid hormone also support this conclusion. Immunochemical quantification of CA III and measurement of CA activity were performed in skeletal muscles of defined fiber-type composition from rats that were rendered hyperthyroid by treatment with 3,3',5-triiodo-L-thyronine. These experiments revealed that CA activity and CA III content are deinduced in the soleus muscle (primarily type I fibers) and induced in the superficial vastus lateralis muscle (primarily type IIb), whereas no changes were detected in the tibialis anterior muscle (primary type IIa). These results show that the modulation of CA III by thyroid hormone in rat skeletal muscle is not limited to the slow-twitch soleus muscle and that the amplitude and direction of this modulation are directly related to the initial fiber-type composition of the skeletal muscle.     

Purification and properties of pig muscle carbonic anhydrase III

Pig muscle carbonic anhydrase III (carbonate hydro-lyase, EC 4.2.1.1) has been isolated and purified to homogeneity with chromatographic techniques. It has been found to be a 30 kDa protein displaying the same three activities (CO2 hydratase, acetate esterase, p-nitrophenyl phosphatase) previously described for the rabbit muscle isoenzyme, including the phosphatase activity not seen in the erythrocyte isoenzymes. The turnover numbers of the three activities are of the same order of magnitude as previously reported for rabbit muscle carbonic anhydrase III. Km and Vmax for the pig muscle CO2 hydratase activity were found to be 83 mM and 6000 s-1, respectively. The extinction coefficient at 280 nm (1 cm light path) is 22.2 for a 1% solution. Five half-cystine residues determined by performic acid oxidation are free for reaction with p-mercuribenzoate but only four are accessible to titration with dithiobisnitrobenzene. The amino acid composition of the pig muscle isoenzyme III has a high level of homology compared with that of rabbit and bovine muscle carbonic anhydrases III.     

Kinetics of CO2 excretion and intravascular pH disequilibria during carbonic anhydrase inhibition

Cardenas, Victor, Jr., Thomas A. Heming, and Akhil Bidani.Kinetics of CO₂ excretion andintravascular pH disequilibria during carbonic anhydrase inhibition.J. Appl. Physiol. 84(2): 683-694, 1998.Inhibition of carbonic anhydrase (CA) activity (activity in redblood cells and activity available on capillary endothelium) results indecrements in CO₂ excretion(CO₂) and plasma-erythrocyteCO₂--H⁺disequilibrium as blood travels around the circulation. To investigate the kinetics of changes in blood PCO₂and pH during progressive CA inhibition, we used our previouslydetailed mathematical model of capillary gas exchange to analyzeexperimental data of CO₂ and blood-gas/pH parameters obtained from anesthetized, paralyzed, andmechanically ventilated dogs after treatment with acetazolamide (Actz,0-100 mg/kg iv). Arterial and mixed venous blood samples werecollected via indwelling femoral and pulmonary arterial catheters, respectively. Cardiac output was measured by thermodilution. End-tidal PCO₂, as a measure of alveolarPCO₂, was obtained from continuousrecords of airway PCO₂ above thecarina. Experimental results were analyzed with the aid of amathematical model of lung and tissue-gas exchange. Progressive CAinhibition was associated with stepwise increments in the equilibratedmixed venous-alveolar PCO₂ gradient(9, 19, and 26 Torr at 5, 20, and 100 mg/kg Actz, respectively). Themaximum decrements in CO₂were 10, 24, and 26% with 5, 20, and 100 mg/kg Actz, respectively,without full recovery ofCO₂ at 1 h postinfusion. Equilibrated arterial PCO₂overestimated alveolar PCO₂, andtissue PCO₂ was underestimated by themeasured equilibrated mixed venous bloodPCO₂. Mathematical model computations predicted hysteresis loops of the instantaneousCO₂--H⁺relationship and in vivo bloodPCO₂-pH relationship due to thefinite reaction times forCO₂--H⁺reactions. The shape of the hysteresis loops was affected by the extentof Actz inhibition of CA in red blood cells and plasma.