Structure of cobalt carbonic anhydrase complexed with bicarbonate.

The three-dimensional structure of a complex between catalytically active cobalt(II) substituted human carbonic anhydrase II and its substrate bicarbonate was determined by X-ray crystallography (1.9 A). One water molecule and two bicarbonate oxygen atoms are found at distances between 2.3 and 2.5 A from the cobalt ion in addition to the three histidyl ligands contributed by the peptide chain. The tetrahedral geometry around the metal ion in the native enzyme with a single water molecule 2.0 A from the metal is therefore lost. The geometry is difficult to classify but might best be described as distorted octahedral. The structure is suggested to represent a water-bicarbonate exchange state relevant also for native carbonic anhydrase, where the two unprotonized oxygen atoms of the substrate are bound in a carboxylate binding site and the hydroxyl group is free to move closer to the metal thereby replacing the metal-bound water molecule. A reaction mechanism based on crystallographically determined enzyme-ligand complexes is represented.     

Direct extracellular interaction between carbonic anhydrase IV and the human NBC1 sodium/bicarbonate co-transporter

Sodium/bicarbonate co-transporters (NBC) are crucial in the regulation of intracellular pH (pH(i)) and HCO(3)(-) metabolism. Electrogenic NBC1 catalyzes HCO(3)(-) fluxes in mammalian kidney, pancreas, and heart cells. Carbonic anhydrase IV (CAIV), which is also present in these tissues, is glycosylphosphatidyl inositol-anchored to the outer surface of the plasma membrane where it catalyzes the hydration-dehydration of CO(2)/HCO(3)(-). The physical and functional interactions of CAIV and NBC1 were investigated. NBC1 activity was measured by changes of pH(i) in NBC1-transfected HEK293 cells subjected to acid loads. Cotransfection of CAIV with NBC1 increased the rate of pH(i) recovery by 44 +/- 3%, as compared to NBC1-alone. In contrast, CAIV did not increase the functional activity of G767T-NBC1 (mutated on the fourth extracellular loop (EC4) of NBC1), and G767T-NBC1, unlike wild-type NBC1, did not interact with CAIV in glutathione-S-transferase pull-down assays. This indicates that G767 of NBC1 is directly involved in CAIV interaction. NBC1-mediated pH(i) recovery rate after acid load was inhibited by 40 +/- 7% when coexpressed with the inactive human CAII mutant, V143Y. V143Y CAII competes with endogenous CAII for interaction with NBC1 at the inner surface of the plasma membrane, which indicates that NBC1/CAII interaction is needed for full pH(i) recovery activity. We conclude that CAIV binds EC4 of NBC1, and this interaction is essential for full NBC1 activity. The tethering of CAII and CAIV close to the NBC1 HCO(3)(-) transport site maximizes the transmembrane HCO(3)(-) gradient local to NBC1 and thereby activates the transport rate.     

The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl^–/HCO ₃ ^– exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl^–/HCO ₃ ^– exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl^–/HCO ₃ ^– exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CA carbonic anhydrase - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - E ₀ total concentration of free enzyme - i fractional inhibition of enzyme activity - IU international units - K ₁ inhibition constant - K _M Michaelis constant - NBT nitro blue tetrazolium - NCP nitrocellulose paper - RBC red blood cell - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max maximal velocity of reaction     

Cyclic AMP effects on chloride transport and carbonic anhydrase activity in frog skin.

Unidirectional (36Cl) chloride fluxes across isolated and short-circuited frog skin were measured, with both sides bathed in low chloride solution. Transepithelial chloride influx was inhibited by exogenous cAMP as well as by substances enhancing its cellular concentration, such as epinephrine, isoproterenol, and 3-isobutyl-1-methylxanthine (IBMX). Epinephrine and isoproterenol addition resulted in an increase of transepithelial chloride outflux, but exogenous cAMP or IBMX had no significant effect on this unidirectional flux. Phenylephrine had no significant effect on influx or outflux. Carbonic anhydrase (CA) activity in extracts obtained from frog skin epithelium was inhibited by pretreatment with IBMX at 4-5 degrees C and prolonged exposure to cAMP at freezing point. cAMP or IBMX alone had no significant effects on CA activity. This catalytic activity was chloride insensitive and was abolished by 0.1 microM acetazolamide. Results suggest a Cl(-)-HCO3- exchange inhibition by cAMP via carbonic anhydrase inactivation. Chloride outflux stimulation by beta-adrenergic agonists does not seem to depend solely on an increase in cAMP concentration.     

Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II.

Twelve amino acid substitutions of varying size and hydrophobicity were constructed at Val 143 in human carbonic anhydrase II (including Gly, Ser, Cys, Asn, Asp, Leu, Ile, His, Phe and Tyr) to examine the catalytic roles of the hydrophobic pocket in the active site of this enzyme. The CO2 hydrase and p-nitrophenyl acetate (PNPA) esterase activities, the pKa of the zinc-water ligand, the inhibition constant for cyanate (KOCN), and the binding constants for sulfonamide inhibitors were measured for various mutants and correlated with the size and hydrophobicity of the substituted amino acid. The kcat/KM for PNPA hydrolysis and KOCN are linearly dependent on the hydrophobicity of the amino acid at position 143. All of the activities of CAII are decreased by more than a factor of 10(3) when large amino acids (Phe and Tyr) are substituted for Val 143, but the CO2 hydrase activity is the most sensitive to the size and structure of the substituted amino acid. Addition of a single methyl group (V143I) decreases the activity 8-fold, while substitution of valine by tyrosine essentially destroys the enzyme function (kcat/KM for CO2 hydration is decreased by more than 10(5)-fold). KOCN does not increase until Phe is substituted for Val 143, suggesting that the cyanate and CO2 binding sites are not identical. The functional data in conjunction with X-ray crystallographic studies of four of the mutants [Alexander et al., 1991 (following paper in this issue)] allow interpretation of the mutants at a molecular level and mapping of the region of the active site important for CO2 association. The hydrophobic pocket, including residues Val 121 and Val 143, is important for CO2 and PNPA association; if the pocket is blocked, substrates cannot approach the zinc-hydroxide with the correct orientation to react. The interaction between Val 143 and CO2 is relatively weak (less than or equal to 0.5 kcal/mol) and nonspecific; the association site does not tightly hold CO2 in one fixed orientation for reaction with the zinc-hydroxide. This mechanism of catalysis may reflect a decreased requirement for specific orientation by CO2 since it is a symmetrical molecule.     

Evidence against a direct interaction between intracellular carbonic anhydrase II and pure C-terminal domains of SLC4 bicarbonate transporters

Based on solid-phase binding assays with enzyme-linked immunosorbent assay detection, previous investigators suggested that intracellular carbonic anhydrase II (CA II) interacts at high affinity with the C-terminal (Ct) domains of SLC4 bicarbonate-transport proteins, expressed as glutathione S-transferase (GST) fusion proteins, to form functional HCO3- metabolons. Here we re-evaluated this protein-protein interaction using two solid-phase binding assays. We first compared the ability of the Ct domain of three SLC4 transporters, SLC4-A1 (AE1), SLC4-A4 (NBCe1), and SLC4-A8 (NDCBE), to bind immobilized CA II, using enzyme-linked immunosorbent assay detection. We found that when expressed as GST fusion proteins, all three bind to CA II (Kd 300-600 nM) better than does pure GST. However, we detected no binding of pure SLC4-Ct peptides to immobilized CA II. Second, we reversed assay orientation by immobilizing the SLC4-Ct fusion proteins or peptides. We found that more CA II binds to GST than to any of the three GST-SLC4-Ct fusion proteins. Furthermore, we detected no binding of CA II to any of the immobilized pure SLC4-Ct peptides. Finally, we used surface plasmon resonance to detect possible rapid interactions between CA II and the pure peptides. Although we detected acetazolamide binding to immobilized CA II and specific antibodies binding to immobilized SLC4-Ct peptides, we detected no binding of CA II to immobilized SLC4-Ct or vice versa. Thus, although an HCO3 metabolon may exist, CA II cannot bind directly to pure SLC4-Ct peptides and can bind to GST-SLC4-Ct fusion proteins only when the CA II is immobilized and the fusion protein is soluble, and not vice versa.     

The functional and physical relationship between the DRA bicarbonate transporter and carbonic anhydrase II

COOH-terminal cytoplasmic tails ofchloride/bicarbonate anion exchangers (AE) bind cytosolic carbonicanhydrase II (CAII) to form a bicarbonate transport metabolon, amembrane protein complex that accelerates transmembrane bicarbonateflux. To determine whether interaction with CAII affects thedownregulated in adenoma (DRA) chloride/bicarbonate exchanger, anionexchange activity of DRA-transfected HEK-293 cells was monitored byfollowing changes in intracellular pH associated with bicarbonatetransport. DRA-mediated bicarbonate transport activity of 18 ± 1 mM H⁺ equivalents/min was inhibited 53 ± 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by themembrane-impermeant carbonic anhydrase inhibitor,1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactiveCAII mutant, V143Y, reduced AE1 transport activity by 61 ± 4%without effect on DRA transport activity (105 ± 7% transport activity relative to DRA alone). We conclude that cytosolic CAII isrequired for full DRA-mediated bicarbonate transport. However, DRAdiffers from other bicarbonate transport proteins because its transportactivity is not stimulated by direct interaction with CAII.

     

Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.     

Functional involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane.

Carbonic anhydrase activity was demonstrated in the chick-embryonic chorioallantoic membrane and was correlated with the Ca2+-transport activity of the membrane. It is inhibited by sulphonamides and is expressed in the chorioallantoic membrane in an age-dependent fashion during embryonic development. Ca2+ uptake by the chorioallantoic membrane in vivo also increases in a similar age-dependent manner. The temporal increase in these activities is coincident with calcium deposition in the embryonic skeleton. Incubation of the chorioallantoic membrane in ovo with sulphonamides specifically inhibits both the carbonic anhydrase and the Ca2+ uptake activities of the membrane in vivo. Enzyme histochemistry revealed the carbonic anhydrase activity is localized in the Ca2+-transporting ectodermal cells of the chorioallantoic membrane. These results, taken together, indicate that carbonic anhydrase may be functionally important in the Ca2+-transport activity of the chorioallantoic membrane.     

The extracellular component of a transport metabolon. Extracellular loop 4 of the human AE1 Cl-/HCO3- exchanger binds carbonic anhydrase IV

Cytosolic carbonic anhydrase II (CAII) and the cytoplasmic C-terminal tails of chloride/bicarbonate anion exchange (AE) proteins associate to form a bicarbonate transport metabolon, which maximizes the bicarbonate transport rate. To determine whether cell surface-anchored carbonic anhydrase IV (CAIV) interacts with AE proteins to accelerate the bicarbonate transport rate, AE1-mediated bicarbonate transport was monitored in transfected HEK293 cells. Expression of the inactive CAII V143Y mutant blocked the interaction between endogenous cytosolic CAII and AE1, AE2, and AE3 and inhibited their transport activity (53 +/- 3, 49 +/- 10, and 35 +/- 1% inhibition, respectively). However, in the presence of V143Y CAII, expression of CAIV restored full functional activity to AE1, AE2, and AE3 (AE1, 101 +/- 3; AE2, 85 +/- 5; AE3, 108 +/- 1%). In Triton X-100 extracts of transfected HEK293 cells, resolved by sucrose gradient ultracentrifugation, CAIV recruitment to the position of AE1 suggested a physical interaction between CAIV and AE1. Gel overlay assays showed a specific interaction between CAIV and AE1, AE2, and AE3. Glutathione S-transferase pull-down assays revealed that the interaction between CAIV and AE1 occurs on the large fourth extracellular loop of AE1. We conclude that AE1 and CAIV interact on extracellular loop 4 of AE1, forming the extracellular component of a bicarbonate transport metabolon, which accelerates the rate of AE-mediated bicarbonate transport.     

Quantitative assessment of intrinsic carbonic anhydrase activity and the capacity for bicarbonate oxidation in photosystem II

On the basis of equilibrium isotopic distribution experiments using (18)O-labeled water, it is generally accepted that water is the sole substrate for O(2) production by photosystem II (PSII). Nevertheless, recent studies indicating a direct interaction between bicarbonate and the donor side of PSII have been used to hypothesize that bicarbonate may have been a physiologically important substrate for O(2) production during the evolution of PSII [Dismukes, G. C., Klimov, V. V., Baranov, S. V., Kozlov, Y. N., DasGupta, J., and Tyryshikin, A. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2170-2175]. To test out this hypothesis and to determine whether contemporary oxygenic organisms have the capacity to oxidize bicarbonate, we employed special rapid-mixing isotopic experiments using (18)O/(13)C-labeled bicarbonate to quantify the inherent carbonic anhydrase activity in PSII samples and the potential flux of oxygen from bicarbonate into the photosynthetically produced O(2). The measurements were made on PSII samples prepared from spinach, Thermosynechococcus elongatus, and Arthrospira maxima. For the latter organism, a strain was used that grows naturally in an alkaline, high (bi)carbonate soda lake in Africa. The results reveal that bicarbonate is not the substrate for O(2) production in these contemporary oxygenic photoautotrophs when assayed under single turnover conditions.     

Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters

Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO₂-HCO₃^– conversions and coupled HCO₃^– transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl^–/HCO₃^– exchanger AE2 or the putative Cl^–/HCO₃^– exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 ± 7% and also activated transport mediated by AE1 and AE3 (32 ± 10 and 37 ± 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl^–/HCO₃^– exchangers to form a bicarbonate transport metabolon. SLC4; SLC26; bicarbonate transport metabolon     

Thermodynamic parameters of the interaction between Co(II) bovine carbonic anhydrase and anionic inhibitors.

The pH dependence of the apparent affinity constants of perchlorate for cobalt(II)bovine carbonic anhydrase II has been measured by electronic absorption spectroscopy. The obtained data have been analyzed in terms of the ionization of two acidic groups of CoBCAII, and the affinity of perchlorate for the two water-containing species of the enzyme have been estimated. Furthermore, the affinity constants of nitrate, perchlorate, and azide for CoBCAII in the temperature range 5 degrees C-30 degrees C have been determined by spectrophotometric titrations at pH 7. The affinity constants for these ligands decrease with increasing temperatures. The temperature dependence of binding was used to estimate the enthalpy and entropy parameters for the formation of the corresponding 1:1 adducts. The obtained results indicate that binding of these anions to the cobalt enzyme is an enthalpy driven process which is opposed by a moderate entropy change.     

Bicarbonate transport and extracellular carbonic anhydrase in marine diatoms

In this article, we present new laboratory results examining the relative importance of HCO⁻₃ transport and extracellular carbonic anhydrase (eCA) in 17 marine diatom species. We observed significant variability in both HCO⁻₃ transport and eCA expression across a range of diatom species with different cell morphologies. All species we examined took up HCO⁻₃ through a direct transport mechanism, with the fraction of HCO⁻₃ transport ranging from 40 to 95% of total C uptake. eCA expression also varied significantly, with catalytic enhancement factors ranging approximately 10-fold among species. There was a significant positive correlation between HCO⁻₃ transport and eCA expression among the test species. However, neither HCO⁻₃ transport nor eCA expression was significantly correlated to cell growth rates or surface area to volume ratios. We did observe weak positive trends between the ratio of C demand:supply and HCO⁻₃ utilization/eCA expression, but these were not statistically significant. We are thus unable to provide a mechanistic explanation for the apparent variability in HCO⁻₃ transport and eCA expression in marine diatoms. This variability may, nonetheless, have important implications for the physiological ecology of oceanic diatoms.     

Carbon isotope effect on dehydration of bicarbonate ion catalyzed by carbonic anhydrase

The carbon-13 kinetic isotope effect on the dehydration of HCO3- by bovine carbonic anhydrase has been measured. To accomplish this, bicarbonate was added to a buffer solution at pH 8 containing carbonic anhydrase under conditions where purging of the product CO2 from the solution is rapid. Measurement of the isotopic composition of the purged CO2 as a function of the concentration of carbonic anhydrase permits calculation of the isotope effect on the enzymic reaction. The isotope effect on the dehydration is k12/k13 = 1.0101 +/- 0.0004. This effect is most consistent with a ping-pong mechanism for carbonic anhydrase action, in which proton transfer to or from the enzyme occurs in a step separate from the dehydration step. Substrate and product dissociation steps are at least 2-3-fold faster than the hydration/dehydration step.