CO2 Hydration Reaction Research Articles

MPV004 Molecular investigations on the intracellular life cycle of Burkholderia pseudomallei

We report the cloning, purification and characterization of BpsβCA, a β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, the etiological agent of melioidosis, and compare its activity and inhibition with those of the γ-CA from the same organism, BpsγCA, recently investigated by our groups. BpsβCA showed a significant catalytic activity for the physiologic, CO2 hydration reaction, with the following kinetic parameters, kcat of 1.6 × 105 s−1 and kcat/Km of 3.4 × 107 M−1 × s−1. The inhibition of BpsβCA with a group of anions and small molecules was also investigated. The best inhibitors were sulfamide, sulfamic acid and phenylarsonic acid, which showed KIs in the range of 83–92 µM, whereas phenylboronic acid, fluoride, cyanide, azide, bisulfite, tetraborate, perrhenate, perruthenate, peroxydisulfate, perchlorate, tetrafluoroborate, fluorosulfonate and hexafluorophosphate showed KIs > 100 mM. Other inhibitors of this new enzyme were bicarbonate, trithiocarbonate, some complex inorganic anions and N,N-diethyldithiocarbamate, which had inhibition constants of 0.32–8.6 mM. As little is known of the life cycle and virulence of this bacterium, this type of study may bring information of interest for the development of novel strategies to fight bacterial infection and drug resistance to commonly used antibiotics.

Novel gene products associated with NdhD3/D4-containing NDH-1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942.

Cyanobacteria possess light-dependent CO2 uptake activity that results in the net hydration of CO2 to HCO3- and may involve a protein-mediated carbonic anhydrase (CA)-like activity. This process is vital for the survival of cyanobacteria and may be a contributing factor in the ecological success of this group of organisms. Here, via isolation of mutants of Synechococcus sp. PCC7942 that cannot grow under low-CO2 conditions, we have identified two novel genes, chpX and chpY, that are involved in light-dependent CO2 hydration and CO2 uptake reactions; co-inactivation of both these genes abolished both activities. The function and mechanism of the CO2 uptake systems supported by each chp gene product differs, with each associated with functionally distinct NAD(P)H dehydrogenase (NDH-1) complexes. The ChpX system has a low affinity for CO2 and is dependent on photosystem I cyclic electron transport, whereas the inducible ChpY system has a high affinity for CO2 and is dependent on linear electron transport. We believe that ChpX and ChpY are involved in a unique, net hydration of CO2 to HCO3-, that is coupled electron flow within the NDH-1 complex on the thylakoid membrane.

Facilitated Transport of Carbon Dioxide Through an Immobilized Liquid Membrane of Aqueous Carbonate Solution with Additives

An experimental study has been carried out to investigate the characteristics of facilitated transport of CO2 through hydrophilic polymeric membranes with an immobilized aqueous potassium carbonate solution. The rate of permeation for CO2 increased with increasing K2CO3 concentration, decreasing membrane thickness, and decreasing CO2 partial pressure. The permeation rate for CO2 was predicted by a theoretical model based on mass transfer accompanied by a reversible chemical reaction, such as hydration reaction of CO2, reaction of CO2 with OH−, or dissociation of HCO− 3. The permeation rate for CO2 increased by adding K2SO3, PEG, or DBC to the aqueous K2CO3 solution. By assuming that these additives enhanced the hydration reaction of CO2, the forward-reaction rate constant was estimated by fitting to the experimental R A . The values estimated thus were 1.35, 0.98, and 0.56 s−1, for K2SO4, polyethyleneglycol, and Dibenzo-18-crown-6, respectively. These values amount to 15–36 times as large as those without additives.

Crystallographic studies of azide binding to human carbonic anhydrase II.

The crystal structures of human carbonic anhydrase II (CAII) at pH 5.7 and 8.0 have been determined at 0.21-nm resolution in the presence of 20 mM azide, which is a noncompetitive inhibitor of the CAII-catalyzed CO2 hydration reaction. Although azide often facilitates the crystallization of CAII and its variants, this small anion does not cause any significant structural changes in the enzyme active site or in the overall protein structure, and zinc coordination remains tetrahedral over the pH range 5.7-8.0. Importantly, the binding of azide at pH 8.0 has implications for the zinc-binding mode of the catalytic product, bicarbonate ion. Since azide is a competitive inhibitor of the reverse reaction of bicarbonate dehydration, and since the zinc-bound azide nitrogen makes a non-hydrogen-bonded van der Waals contact with the hydroxyl group of Thr199, it is possible that a zinc-bound bicarbonate oxyanion could likewise make a non-hydrogen-bonded, van der Waals contact with the hydroxyl group of Thr199. Therefore, the donation of a hydrogen bond to Thr199 is not absolutely required for anion binding to tetracoordinate zinc.

The effect of bile salts on carbonic anhydrase

Bile salts are potent inhibitors of bovine carbonic anhydrase and human carbonic anhydrase I and human carbonic anhydrase II. To further characterize the binding of bile salts to carbonic anhydrase, rate constants for the CO2 hydration reaction in the presence of deoxycholate, cholate, glycocholate and taurocholate were determined using stop-flow experiments. Values for the Michaelis-Menton dissociation constant for bovine carbonic anhydrase, human carbonic anhydrase I and human carbonic anhydrase II were found to be 5.2, 9.2 and 13.2 mmol/L, respectively. The inhibition constant values for the various bile salts tested ranged from 0.1 to 1 mmol/L for bovine carbonic anhydrase, 1.6 to 2.4 mmol/L for human carbonic anhydrase I and 0.09 to 0.7 mmol/L for human carbonic anhydrase II. Our results suggest a mechanism of noncompetitive carbonic anhydrase inhibition for bile salts. Bile-salt binding to carbonic anhydrases as measured by scanning molecular sieve chromatography resulted in an increase in partition radius, molecular volume and surface area. The partition radius increased from 24 A to 28 A in the presence of 2.5 mmol/L sodium deoxycholate at critical micelle concentration. As determined by sedimentation equilibrium measurements, approximately 1 gm of carbonic anhydrase will bind 0.03 gm of deoxycholate, suggesting three to six binding sites for bile salt on the carbonic anhydrase molecule. The conformational changes and inhibition of carbonic anhydrases resulting from bile-salt binding may be important to the regulation of enzymatic activity in tissues along the enterohepatic circulation; by limiting bicarbonate availability this interaction may also contribute to the metabolic derangements seen in patients with cholestatic liver disease.

String model for the rate constant of nonadiabatic solvation in the hydration reaction of carbon dioxide

AbstractPerturbation theory for the rate constant of nonadiabatic solvation is presented. Miller's treatment of the RRKM theory is used for the unperturbed rate constant, and thereby the “string model” of chemical reaction is used for the description of the perturbation of solvation. The reaction path under the influence of the external force field is defined as the intrinsic reaction coordinate (IRC) that is treated as a string. The string is thrown in the external static force field that acts as a nonadiabatic source of perturbation. As an application of the present treatment, the effect of the external point charge for hydration reaction of CO2 is calculated as a preliminary model for catalytic activity of the carbonic anhydrase in the tissues and lung capillary vessels.

The interaction between carbon dioxide and ammonia excretion and water pH in fish

The pH of plasma is midway between the pK of the [Formula: see text] and [Formula: see text] reactions. Carbon dioxide excretion across the gill acidifies the water, as CO2 hydrates to form [Formula: see text] and H+ ions. The hydration reaction is catalyzed by carbonic anhydrase in the water boundary layer next to the gills and on the gill epithelium. Acidification of the boundary layer is greater than in the bulk flow, particularly in water of high pH and low buffering capacity. Ammonia excretion will raise water pH but this effect is usually masked by the much larger CO2 excretion. At low water pH, below the pK′ of the CO2 hydration reaction, little [Formula: see text] and H+ will be formed, therefore CO2 will have little effect on downstream water pH. Under these conditions, NH3 excretion will alkalinize water as it passes over the gills. Formation of an acid gill water boundary layer as a result of CO2 excretion will trap NH3 as [Formula: see text] and enhance ammonia transfer. Removal of the acid boundary layer, either by increasing the buffering capacity of the water or by reducing CO2 excretion, decreases ammonia transfer in the isolated head of rainbow trout. Water pH can have a marked effect on CO2 and NH3 gradients across the gills. In alkaline waters, the rise in blood NH3 levels will result in a marked increase in body stores of total ammonia. Tilapia (Oreochromis alcalicus grahami) in Lake Magadi, Kenya, at a water pH of 10, excrete nitrogenous wastes as urea, to avoid the problem of the buildup of toxic levels of ammonia when the fish are exposed to these alkaline conditions.

Mass spectrometric determination of the inorganic carbon species assimilated by photoautotrophic cells of Euphorbia characias L.

The chemical forms of inorganic carbon, CO2 or HCO3-, incorporated during photosynthesis in photoautotrophic Euphorbia characias cell suspension cultures were determined in experiments using 13CO2 and a mass spectrometry technique. From the equations of the CO2 hydration reaction, a kinetic model was first developed, and the effect of photosynthesis on the external CO2 concentration was simulated. It was predicted from this model that CO2 and HCO3- uptakes could be differentiated by recording only the CO2 variation rate in the external medium, successively in absence then in presence of an exogenous carbonic anhydrase activity. The results obtained with either CO2-grown or air-grown photoautotrophic cells were in good agreement with the model and demonstrated that CO2 was the sole species taken up during photosynthesis. In addition no accumulation of inorganic carbon within the cells was observed in the light. Similarly, in dark, CO2 was the only species released by respiration in the external medium.

Delayed ventilatory response to CO2 after carbonic anhydrase inhibition with acetazolamide administration in the anesthetized rat.

In order to estimate to what extent the stimulatory action of CO2 on ventilation is mediated by the formation of H+, we studied effects on the temporal profile of ventilatory response to CO2 of carbonic anhydrase (CA) inhibition with acetazolamide in the halothane anesthetized spontaneously breathing rat. Since hydration reaction of CO2 yielding H+ is delayed by CA inhibition, the time courses of changes in tidal volume (VT), respiratory frequency (f), and minute ventilation (VE) in response to a stepwise increase in end-tidal PCO2 (delta PETCO2 15 mmHg) were compared before (control state) and after i.v. injection of acetazolamide (50 mg/kg) in the hyperoxic condition. In the control state, an increase in VT was significantly slower than that in f, and the mean response half-times (T1/2) for the increase in VT, f, and VE were 50.6, 18.1, and 31.0 s, respectively. After acetazolamide administration, responses to CO2, especially f-response and consequently VE-response became much slower, and the T1/2 for VT, f, and VE were 67.9, 55.0, and 63.0 s, respectively. The delay in VT-response was not statistically significant. The magnitude of increase in VE in the steady state hypercapnic stimulation was almost the same before and after acetazolamide administration. The results suggest that a rapid increase in f during CO2 inhalation occurs predominantly through an increase in H+ produced by hydration of CO2 with CA, whereas VT-response may occur without involvement of this process. The different time courses of VT- and f-responses and possible effects of molecular CO2 and/or H+ on the regulatory mechanisms for ventilatory pattern were discussed.

Molecular basis of ionic strength effects: interaction of enzyme and sulfate ion in CO2 hydration and HCO3- dehydration reactions catalyzed by carbonic anhydrase II.

CO2 hydration and HCO3- dehydration reactions catalyzed by carbonic anhydrase II have been examined at various concentrations of sodium sulfate with a stopped-flow technique. We find that at low ionic strength CO2 hydration and HCO3- dehydration reaction rates remain unaffected by varying the salt concentration at pH higher than 7.0, while the reaction rates decrease with increasing ionic strength at low pH. For CO2 hydration reactions, salt effects reside only in the kcat term, not in the Km term, whereas for HCO3- dehydration reactions, salt effects reside only in the Km term, not in the kcat term. In this regime, the salt concentration dependence of the turnover rate for CO2 hydration at low pH is attributed to an electrostatic effect on the ionization constants of the enzyme and/or enzyme-substrate complex, which in turn affect the pH profile of kcat. The rates of the bimolecular interaction between the uncharged CO2 molecule and carbonic anhydrase II at high pH are unaffected by low salt concentration while the rates of the bimolecular interaction of HCO3- with enzyme at low pH decrease with increasing salt concentration, consistent with a negative salt effect on an electrostatically enhanced diffusion of the negatively charged substrate to the positively charged active site. These bimolecular reactions between enzyme and substrate at low ionic strength obey rate equations derived from the Debye-Hückel limiting law and the transition-state theory. Simple linear relationships between the logarithm of the catalytic constant, log kdenz, and the square root of the ionic strength were established.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat lung carbonic anhydrase: activity, localization, and isozymes.

Carbonic anhydrase activity in rat lungs perfused free of blood was localized by homogenization of the tissue followed by differential centrifugation. Four fractions were obtained from the homogenate, a cell debris pellet with a mitochondrial pellet and a microsomal pellet with a clear cytosol supernatant. The last named fraction contained 67% of the total enzyme activity; the cell debris contained 18%, and the mitochondrial and microsomal contained 8 and 7%, respectively. Of the 33% of enzyme activity associated with the pellet fraction, 25% could be experimentally defined as membrane associated by its solubilization with 0.3 M tris-(hydroxymethyl) aminoethane sulfate buffer. The remainder was defined as membrane bound. Purification of the soluble carbonic anhydrase from the lung yielded two isozymes with electrophoretic and inhibitor sensitivities apparently identical with the blood isozymes. Hemoglobin analysis showed that the lung isozymes could not have included more than 0.03% enzyme from blood contamination. The carbonic anhydrase activity present in the whole rat lung would give an average acceleration of the CO2 hydration reaction under physiological conditions over the uncatalyzed rate of 122, sufficient to maintain equilibration between CO2 and plasma HCO3- during blood transit of the lung. If the membrane-associated activity is mostly on the plasma membrane of the endothelial cells and available to the capillary blood, it would be sufficient to give this acceleration. We suggest that the possible source of this membrane-associated activity might be adsorption from the blood of carbonic anhydrase liberated by erythrocyte lysis.

Inhibition of carbonic anhydrase by plasma of dogs and rabbits.

Carbonic anhydrase (CA) activity was measured by the Bowes-Davis technique in diluted hemolysates of dog erythrocytes, rabbit erythrocytes, and dog lung tissue homogenates. Plasma (from the same animal) inhibited the CA activity in each case. For 1:16,700 dilution of dog erythrocytes, the CA catalyzed the CO2 hydration reaction by 5.3 +/- 0.4-fold above the uncatalyzed rate, and half that activity was inhibited by plasma concentrations of 0.44 +/- 0.05%. Similar rabbit CA concentrations were inhibited by plasma concentrations of 1.02 +/- 0.24%. CA from dog lung tissue homogenate is only partially inhibited by plasma even at high plasma concentrations, suggesting different isozymes, at least one of which is not inhibited by plasma. The results suggest that extrapolating from artificially perfused lungs or histological observations to in vivo conditions may not be valid, and the possibility of inhibition by plasma in at least some species should be considered.

Physical, chemical and biological characteristics* of CO2 gas flux across the air‐water interface

Abstract Carbon dioxide gas flux across the air‐water interface is most often treated as a ‘simple’ physical process, primarily responding to wind speed and water temperature. Available experimental data yield an exponential regression equation relating wind speed to the thickness of a stagnant boundary film through which gas diffuses to or from the water. Flux of CO2 is influenced by CO2 hydration reactions in the stagnant boundary layer. High pH and a thick stagnant boundary layer favour chemical enhancement of the CO2 gas flux.The rate of CO2 flux reflects the sum of net organic metabolism plus CaCO3 reactions. Some interesting gas‐flux constraints on the rate of net organic carbon production and on global geochemical cycling of CaCO3 emerge. At high pH (circa 10), the maximum net organic carbon production which can be supported by CO2 flux across the air‐water interface is about 0.06 mol C m&2 d&1. On a global scale, organic C, not atmospheric C, appears to account for the ‘CO2’ term in the classical CaCO3 dissolution‐precipitation reaction.

ELECTROKINETIC EFFECTS: CARBONIC ANHYDRASE AND CO2ELECTRODE DYNAMICS

It is well known that the dynamic response rate of the Severinghaus-type CO2 electrode is improved significantly in some cases by addition of the enzyme carbonic anhydrase to the electrode assembly. Hysteresis in the response rate also is reduced. Experimental data and modelling results indicate that catalysis of the CO2 hydration reaction in the bulk of the bicarbonate layer (the Nernst film) is not responsible for the improved response behavior, Evidence is presented to show that catalysis in the electrostatic double layer region at the glass electrode surface is a possible explanation. This proposed phenomenon may have widespread implications for the optimal design of analytical devices, commercial processes involving electrochemical phenomena, and may also provide insight into electrobiologi-cal processes.

Electric potentials due to CO2 diffusion in myoglobin solutions.

The passage of CO2 through water layers is attended by concurrent transport of (bi)carbonate. Since both bicarbonate and carbonate are charged molecules, their diffusion may evoke electric potentials and thus there will be interaction with all other ions present in the solution. Such effects have been described in the literature for weak acids and protein solutions of hemoglobin and albumin (Stroeve et al., 1985). Here, we present measurements of electric potential difference across layers containing 1.5 mmol/l myoglobin at 25 degrees C and various amounts of Na+ ions, where chemical equilibrium for the hydration reaction of CO2 was achieved by adding carbonic anhydrase. Voltages measured ranged from up to 5 mV at zero cation concentration to 0.7 mV around 100-150 mmol/l [Na+]. Contrary to hemoglobin and albumin, these voltages are lower, and even much lower, than theoretical predictions.

Measurement of pulmonary venous and arterial pH oscillations in dogs using catheter tip pH electrodes.

Respiratory pH oscillations were studied in situ in the aorta and superior vena cava of rabbits, and in the pulmonary vein and aorta of open-chested dogs, using catheter tip pH electrodes. Preliminary experiments on two spontaneously breathing rabbits showed pH oscillations of 0.006-0.010 pH units in the ascending aorta at a respiratory rate of 40/min. No oscillations were apparent in the superior vena cava. In anaesthetized, ventilated dogs, the delay from the upstroke in airway pressure to the onset of the alkaline limb of the pH oscillation was 1.15 +/- 0.9 s in the pulmonary vein and 4.1 +/- 0.1 s in the descending aorta (mean + S.E. of mean, n = 28 tracings in four dogs). Pulmonary venous oscillations were equal to or larger in amplitude than those in the aorta. Aortic oscillations became proportionately smaller at shorter respiratory cycle lengths, although both were related to tidal volume. Stop flow experiments were performed using a pH electrode placed in situ in the left atrium inside a wide-bore tube. A small fall in pH occurred when flow was stopped (-0.0036 +/- 0.0012, mean +/- S.E. of mean, n = 22 recordings in four dogs). The findings were not dependent upon the phase of the oscillation at which flow was stopped. Mean plasma free haemoglobin was very low (1.6 mg/dl), indicating minimal haemolysis of the blood withdrawn past the electrode. It is concluded that the plasma CO2 hydration reaction is virtually at equilibrium by the time blood reaches the pulmonary vein.

FACILITATED TRANSPORT OF CARBON DIOXIDE: A REVIEW

Abstract The effect of reversible chemical reaction upon the diffusion of carbon dioxide in thin liquid film membranes is reviewed. Particular emphasis is placed upon the hydration reaction of CO2 and catalysis thereof. Theoretical analysis is developed for equilibrium, near-equilibrium and near-diffusion regimes. The important effects of weak acid buffers, proteins and amines upon CO2 transport are also discussed, as well as the significance of diffusion potentials, particularly in the case of protein systems. Results of experimental investigations of facilitated CO2 transport are reviewed, as well as general aspects of CO2 transport in biological systems and absorbers.

Theoretical model for exploration of catalytic activity of enzymes and design of new catalysts: CO2 hydration reaction

AbstractThe quantum chemical model for predicting the optimal molecular environment exerting the highest catalytic activity on transition complex has been proposed. It was applied for the CO2 hydration reaction at the nonempirical LCAO MO SCF level. The possible use of the resulting optimal charge distribution of environment for design of new synthetic catalysts and explanation of the carbonic anhydrase action was discussed. In addition, a simplified approach based on the difference electrostatic molecular potentials evaluated within the many center multipole expansion is presented. It may enable us to perform corresponding estimations even by means of a programmable calculator.

Theoretical studies on substrate binding to the active site of carbonic anhydrase

AbstractThe role of some amino acids and metal ions in the catalytic activity of carbonic anhydrase (carbonate dehydratase EC 4.2.1.1) has been investigated. The additional stabilization of the transition state complex was used as the qualitative measure of the effect of molecular surrounding on CO2 hydration reaction calculated within the approximate CNDO/2 approach. The effect of the molecular environment has been simulated by inclusion into the SCF LCAO MO Hamiltonian, a term representing the interaction with a set of point charges and atomic dipoles centered on experimentally determined position of Zn2+ ion and all atoms of histidine 94, 96, 119 and threonine 199, 200 (or histidine 200 in the case of carbonic anhydrase B). The possible molecular mechanism of CO2 hydration inside the active site has been also discussed.

H+ transport from CNS in hypercapnia and regulation of CSF [HCO3-

CSF HCO3- increases more than plasma HCO3- in hypercapnia, and there are at least two sources for the CSF HCO3- increase--one derived from the simultaneous increase in plasma HCO3-, and the other, HCO3-formed from hydration of CO2 in the choroid plexus and glia and susceptible to inhibition by acetazolamide (J. Appl. Physiol. 38: 504-512, 1975). It was proposed that the H+ formed in the CNS in CO2 hydration is actively exchanged for plasma Na+ utilizing the Na-K ATPase pump. H+ transport from the CNS was therefore studied in four groups of dogs breathing 5% CO2 at constant VA for 4 h with repeated injections of saline, acetazolamide 5 mg/ml, ouabain 0.1 mg/ml, and acetazolamide and ouabain together into lateral cerebral ventricles. Arterial HCO3-increased 2.5 meq/l at 4 h of hypercapnia in all groups. CSF HCO3-increased 5.8 meq/l in the saline-injected animals, but it increased only about 2 meq/l and equaled plasma HCO3- rise in the other three groups. Therefore CNS HCO3- formation in hypercapnia can be blocked by inhibiting the CO2 hydration reaction with acetazolamide or by blocking H+ removal by inhibiting Na-K ATPase with ouabain. The data support the thesis of active H+ removal from the CNS in exchange for plasma Na+ in hypercapnia.