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-.     

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.     

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.     

Periplasmic expression of carbonic anhydrase in Escherichia coli: A new biocatalyst for CO2 hydration

Carbonic anhydrase is a valuable and efficient catalyst for CO₂ hydration. Most often the free enzyme is employed which complicates catalyst recycling, and can increase cost due to the need for protein purification. Immobilization of the enzyme may address these shortcomings. Here we report the development of whole‐cell biocatalysts for CO₂ hydration via periplasmic expression of two forms of carbonic anhydrase in Escherichia coli using two different targeting sequences. The enzymatic turnover numbers (k_cat) and catalytic efficiencies (k_cat/K_M) were decreased by an order of magnitude as compared to the free soluble enzyme, indicating the introduction of transport limitations. However, the thermal stabilities were improved for most configurations (>88% activity retention up to 95°C for three of four whole‐cell biocatalysts), operational stabilities were more than satisfactory (100% retention after 24 h of use for all four whole‐cell biocatalysts), and CO₂ hydration was significantly enhanced relative to the uncatalyzed reaction (～50–70% increase in CaCO₃ precipitate formed). A significant advantage of the whole‐cell approach is that protein purification is no longer necessary, and the cells can be easily separated and recycled in future applications including biofuel production, biosensors, and carbon capture and storage. Biotechnol. Bioeng. 2013; 110: 1865–1873. © 2013 Wiley Periodicals, Inc.     

Histidine 64 is not required for high CO2 hydration activity of human carbonic anhydrase II

To test the hypothesis that histidine 64 in carbonic anhydrase II has a crucial role as a 'proton shuttle group' during catalysis of CO2-HCO3- interconversion, this residue was replaced by lysine, glutamine, glutamic acid and alanine by site-directed mutagenesis. All these variants turned out to have high CO2 hydration activities. The kcat values at pH 8.8 and 25 degrees C were only reduced by 1.5-3.5-fold compared to the unmodified enzyme. These results show that intramolecular proton transfer via His 64 is not a dominating pathway in the catalytic reaction. The variants also catalyze the hydrolysis of 4-nitrophenyl acetate. The pKa values for the activity-controlling group are between 6.8 and 7.0 for all studied forms of the enzyme except the Glu 64 variant which shows a complex pH dependence with the major pKa shifted to 8.4.     

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.     

Kinetic study of catalytic CO(2) hydration by water-soluble model compound of carbonic anhydrase and anion inhibition effect on CO(2) hydration

A kinetic study of CO(2) hydration was carried out using the water-soluble zinc model complex with water-soluble nitrilotris(2-benzimidazolylmethyl-6-sulfonate) L1S, [L1SZn(OH(2))](-), mimicking the active site of carbonic anhydrase, in the presence and absence of anion inhibitors NCS(-) and Cl(-). The obtained rate constants k(cat) for CO(2) hydration were 5.9x10(2), 1. 7x10(3), and 3.1x10(3) M(-1) s(-1) at 5, 10, and 15 degrees C, respectively: the k(cat)=ca. 10(4) M(-1) s(-1) extrapolated towards 25 degrees C has been the largest among the reported k(cat) using zinc model complexes for carbonic anhydrase. It was also revealed that NCS(-), Cl(-) and acetazolamide play a role of inhibitors by the decrease of k(cat): 7x10(2) and 2x10(3) M(-1) s(-1) for NCS(-) and Cl(-) at 15 degrees C, respectively. The sequence of their magnitudes in k(cat) is Cl(-) approximately acetazolamide>NCS(-), where the sequence Cl(-)>NCS(-) is confirmed for native carbonic anhydrase. The difference of k(cat) or k(obs) between NCS(-) and Cl(-) resulted from that between the stability constants K(st)=2x10(3) for [L1SZn(NCS)](2-) and 1x10(2) M(-1) for [L1SZnCl](2-) in D(2)O: for water-insoluble tris(2-benzimidazolylmethyl)amine L1, K(st)=1.8x10(4) for [L1Zn(NCS)](2-) and 1.5x10(3) M(-1) for [L1ZnCl](2-)in CD(3)CN/D(2)O (50% v/v). The crystal structure of anion-binding zinc model complexes [L1Zn(OH(2))](0.5)[L1ZnCl](0.5) (ClO(4))(1.5) 1(0.5)2(0.5)(ClO(4))(1.5) was revealed by X-ray crystallography. The geometry around Zn(2+) in 1 and 2 was tetrahedrally coordinated by three benzimidazolyl nitrogen atoms and one oxygen atom of H(2)O, or Cl(-).     

Nucleophilic reaction by carbonic anhydrase model zinc compound: characterization of intermediates for CO2 hydration and phosphoester hydrolysis

The partially hydrophilic and hydrophobic tripodal ligands, tris(hydroxy-2-benzimidazolylmethyl)amine L1h and tris(2-benzimidazolyl)amine L1 were used for the preparation of biomimetic complex of carbonic anhydrase. The CO(2) hydration using [L1hZn(OH)]ClO(4).1.5H(2)O provided the zinc-bound and free HCO(3)(-)s, which were formed by nucleophilic attack of Zn-OH toward CO(2) in dimethyl sulfoxide (DMSO). The phenolic OH in L1h can recognize water molecules through hydrogen bonds to facilitate the collection of the water molecules around a biomimetic zinc compound; the molecular structure of [L1hZn(OH)](+) was revealed. The packing diagram has demonstrated the all the water molecules are hydrogen bonded to each phenolic OH. The nucleophilic attack of zinc-bound OH(-) to substrate is used to catalyze the CO(2) hydration and phosphoester hydrolysis. The carbonic anhydrase model compound [L1Zn(OH(2))](2+) was applied for the hydrolysis of phosphoesters, parathion and bis(p-nitrophenyl)phosphate (BNPP(-)). The low reactivity of [L1Zn(OH)](+) for parathion hydrolysis is attributed to the stability of the intermediate [L1Zn(OP(S)(OEt)(2))](+). Since the structures of the intermediates [L1Zn(OH(2))](BNPP)(2) (1) and [L1Zn(OP(S)(OEt)(2))]ClO(4) (2) formed on the way of hydrolysis are too stable to realize the catalytic cycle and are not active for hydrolysis, carbonic anhydrase model compound [L1Zn(OH(2))](2+) was not suitable for phosphoester hydrolysis; the zinc model compound surrounded by three benzimidazolyl groups is used to have the steric hindrance for bulky substrate, such as parathion and BNPP(-).     

Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )     

Control of flatfish sperm motility by CO2 and carbonic anhydrase

Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25-100 mM HCO3-, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3-. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3(-)-arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/HCO3(-)-dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm.     

The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney

The steady-state kinetic parameters for the hydration of CO₂ catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number k_cat is 6.5 × 10⁵ s^?1 and the Michaelis constant is 7.5 mm for CO₂ hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 10⁵ s^?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 10⁸m^?1s^?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO₂ in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO₂ hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase.     

Topical inhibition of nasal carbonic anhydrase affects the CO2 detection threshold in rats

Previous studies indicate that Long-Evans rats can be operantly trained to discriminate inspired CO(2) concentrations as low as 0.5%. This ability has been proposed to be due to the presence of CO(2)-sensitive olfactory receptors that contain the enzyme carbonic anhydrase (CA). The objectives of the present study were as follows: 1) to determine whether Zucker rats could be operantly conditioned to discriminate low concentrations of CO(2) from control air and 2) to determine the rats' CO(2) detection thresholds before and after nasal perfusion of mammalian Ringers or methazolamide, a CA inhibitor. Rats were operantly trained to discriminate between 25% CO(2) and control air (0% CO(2)) and were then subjected to various CO(2) concentrations (0.5-12.5%) to determine their CO(2) detection thresholds. The average (+/-standard error of mean) baseline CO(2) detection threshold of 7 Zucker rats was 0.48 +/- 0.07% CO(2), whereas the average CO(2) detection thresholds after nasal perfusion of either mammalian Ringers or 10(-2) M methazolamide were 1.41 +/- 0.30% and 5.92 +/- 0.70% CO(2), respectively. The average CO(2) detection threshold after methazolamide was significantly greater (P<0.0001) than the baseline detection threshold. These findings demonstrate that like Long-Evans rats, Zucker rats can be trained to discriminate low concentrations of CO(2) and that inhibition of nasal CA reduces the ability of the rats to detect low concentrations (3.5% and below) but not higher concentrations of CO(2) (12.5%). These results add to the growing evidence that olfactory neurons exhibiting CA activity are CO(2) chemoreceptors sensitive to physiological concentrations of CO(2).     

Activity and stability of immobilized carbonic anhydrase for promoting CO2 absorption into a carbonate solution for post-combustion CO2 capture

An Integrated Vacuum Carbonate Absorption Process (IVCAP) currently under development could significantly reduce the energy consumed when capturing CO2 from the flue gases of coal-fired power plants. The biocatalyst carbonic anhydrase (CA) has been found to effectively promote the absorption of CO2 into the potassium carbonate solution that would be used in the IVCAP. Two CA enzymes were immobilized onto three selected support materials having different pore structures. The thermal stability of the immobilized CA enzymes was significantly greater than their free counterparts. For example, the immobilized enzymes retained at least 60% of their initial activities after 90 days at 50 °C compared to about 30% for their free counterparts under the same conditions. The immobilized CA also had significantly improved resistance to concentrations of sulfate (0.4 M), nitrate (0.05 M) and chloride (0.3 M) typically found in flue gas scrubbing liquids than their free counterparts.