Carbonic anhydrases in plants and algae

Carbonic anhydrases catalyse the reversible hydration of CO₂, increasing the interconversion between CO₂ and HCO₃⁻ + H⁺ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C₃ dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.     

A new affinity gel for the purification of α-carbonic anhdrases

Abstract

The new affinity gel reported in this study was prepared using EUPERGIT C250L as a chromatographic bed material, to which etylenediamine spacer arms were attached to prevent steric hindrance between the matrix and ligand, and to facilitate effective binding of the CA-specific ligand, of the aromatic sulfonamide type for the purification of α-carbonic anhydrases (Cas; EC 4.2.1.1). Indeed, the aminoethyl moieties of the affinity gel were derivatized by reaction with 4-isothiocyanatobenzenesulfonamide, with the formation of a thiourea-based gel, having inhibitory effects against CAs. Both bovine erythrocyte carbonic anhydrase BCA and human (h) erythrocyte CA isoforms I, II (hCA I and II) have been purified from hemolysates, by using this affinity gel. The greatest purification fold and column yields for BCA and for cytosolic (hCA I?+?II) enzymes were of 181-fold (21.07%) and 184-fold (9.49%), respectively. Maximum binding was achieved at 15?°C and I?=?0.3 ionic strength for α-carbonic anhydrases.     

Carbonic anhydrase metabolism is a key factor in the toxicity of CO2 and COS but not CS2 toward the flour beetle Tribolium castaneum [Coleoptera: Tenebrionidae

The analogues carbon dioxide (CO(2)), carbonyl sulfide (COS) and carbon disulfide (CS(2)) have been useful as substrate probes for enzyme activities. Here we explored the affinity of the enzyme carbonic anhydrase for its natural substrate CO(2), as well as COS and CS(2) (1) by in vitro kinetic metabolism studies using pure enzyme and (2) through mortality bioassay of insects exposed to toxic levels of each of the gases during carbonic anhydrase inhibition. Hydrolysis of COS to form hydrogen sulfide was catalysed rapidly showing parameters K(m) 1.86 mM and K(cat) 41 s(-1) at 25 degrees C; however, the specificity constant (K(cat)/K(m)) was 4000-fold lower than the reported value for carbonic anhydrase-catalysed hydration of CO(2). Carbonic anhydrase-mediated CS(2) metabolism was a further 65,000-fold lower than COS. Both results demonstrate the deactivating effect toward the enzyme of sulfur substitution for oxygen in the molecule. We also investigated the role of carbonic anhydrases in CO(2), COS and CS(2) toxicity using a specific inhibitor, acetazolamide, administered to Tribolium castaneum (Herbst) larvae via the diet. CO(2) toxicity was greatly enhanced by up to seven-fold in acetazolamide-treated larvae indicating that carbonic anhydrases are a key protective enzyme in elevated CO(2) concentrations. Conversely, mortality was reduced by up to 12-fold in acetazolamide-treated larvae exposed to COS due to reduced formation of toxic hydrogen sulfide. CS(2) toxicity was unaffected by acetazolamide. These results show that carbonic anhydrase has a key role in toxicity of the substrates CO(2) and COS but not CS(2), despite minor differences in chemical formulae.     

Purification and properties of cyclostome carbonic anhydrase from erythrocytes of hagfish

1. Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been purified from erythrocytes of hagfish (Myxine glutinosa). A single form with low specific CO2 hydration activity was isolated. The purified carbonic anhydrase appeared homogeneous judging from polyacrylamide gel electrophoresis and gel filtration experiments. The protein has a molecular weight of about 29 000, corresponding to about 260 amino acid residues. This molecular weight is in accordance with other vertebrate carbonic anhydrases with the exception of the elasmobranch enzymes, which have Mr 36 000--39 000. 2. The molecular weight obtained for hagfish carbonic anhydrase indicates that a carbonic anhydrase with Mr approx. 29 000 is the ancestral type of the vertebrate enzyme rather than, as in sharks, a heavier carbonic anhydrase molecule. 3. The circular dichroism spectrum may indicate a somewhat different structural arrangement of aromatic amino acid residues in this enzyme than in the mammalian carbonic anhydrases. 4. The enzyme is strongly inhibited by acetazolamide and also to a lesser extent by monovalent anions. 5. Zn2+, which is essential for activity, appears, contrary to other characterized carbonic anhydrases, less strongly bound in the active site of the enzyme.     

Affinity chromatography of carbonic anhydrase

An insoluble support for affinity chromatography of carbonic anhydrase has been prepared by coupling Sulfamylon (p-aminomethylbenzene sulfonamide) to Sepharose 4B. Carbonic anhydrase binds to Sulfamylon-Sepharose very strongly and can be eluted under mild conditions by the addition of enzyme inhibitors. The gel was used to purify carbonic anhydrase from human erythrocytes and to separate isozymes B and C. It was also employed to separate native enzyme from modified carbonic anhydrases. The apoenzyme and the carboxymethyl enzyme of human carbonic anhydrase B were both isolated by this method.     

Mechanistic studies of the astacin-like <Emphasis Type="Italic">Serratia</Emphasis> metalloendopeptidase serralysin: highly active (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism

Serralysin is a bacterial Zn-endopeptidase which has been considered a virulence factor to cause tissue damage and anaphylactic response. It contains a coordinated Tyr that is unique to the astacin-like Zn enzymes. The coordinated Tyr has been proposed to play an important role in the action of this endopeptidase family. Several metal-substituted derivatives of serralysin (including Mn2+, Co2+, Ni2+, Cu2+, and Cd2+ derivatives) are found to exhibit significant activities. Particularly, the Co- and Cu-substituted derivatives exhibit much higher activities than the native serralysin toward the hydrolysis of the tripeptide mimic benzoyl-Arg- p-nitroanilide, i.e., 35 and 49 times higher in k(cat) and 33 and 26 times in k(cat)/ K(m), respectively. Such remarkably higher activities of metal-substituted derivatives, especially the Cu derivative, than that of the native Zn enzyme are rare in the literature, reflecting the uniqueness of this enzyme among all Zn enzymes. The significantly different k(cat) yet similar K(m) values among the several metal derivatives suggests that the metal center is involved in catalysis, but not necessarily in the binding of the substrate, whereas the dramatically different inhibition constants for Arg-hydroxamate binding to the metal-substituted derivatives indicates direct binding of this inhibitor to the metal center. The activity-pH profiles of serralysin and its Co2+ and Cu2+ derivatives and the optical-pH profile of Cu-serralysin have been obtained, in which the decrease in activity at higher pH values was found to be associated with a dramatic increase in the Tyr-to-Cu2+ charge transfer transitions. This observation suggests that the binding of Tyr216 to the metal is inhibitory. A metal-centered mechanism is proposed for serralysin catalysis based on the results presented here, in which the detachment of the coordinated Tyr and formation of a H-bond with the transition-state complex are considered essential for the stabilization of the transition state.     

Carbonic anhydrases of bovine erythrocytes: comparative study of the anhydrase and esterase activity of different forms]

Comparative study of esterase activities (p- and o-nitrophenylacetate) allowed to characterize three groups of bovine erythrocyte carbonic anhydrases:--the first one includes CI, CII (isozyme of CI) and CIr ("artificial" product of CI).--the second one includes native CIv1 and "artificial" CIv1, first conformational variants of CI,--finally CIv2, second "artificial" conformational variant of CI. Possible modifications of the enzyme site between the first and the other enzyme groups are discussed. Except CIv2 of lower activity, all the products have identical carbonic anhydrase activity. The catalytic constants Km ap and kcat ap for hydrolysis of p-nitrophenylacetate have been determined for all enzymes; this study confirms the lower activity of CIv2.     

Studies of Zn(II) and Co(II) complexes of imidazole and n-methylimidazole with regard to the activity related ionization in carbonic anhydrase.

Mixed aquo-N-methylimidazole complexes of Co(II) have been studied as a function of pH to gain a fuller understanding of the metal-binding site in Co(II)-carbonic anhydrase. The inherent affinity of N-methylimidazole for Co(II) has been calculated along with a species distribution for the stepwise addition of ligand to the metal ion. From these studies, it is apparent that the occurrence of Zn(II) rather than Co(II) in native carbonic anhydrase can be explained by the stronger affinity of Zn(II) for imidazole and the preference of Zn(II) for a tetrahedral geometry as offered by the enzyme. Octahedral Co(II) fails to ionize metal bound water. However, at high pH, Co(II)-N-methylimidazole complexes interact directly with the hydroxide ion, generating species with visible spectra very similar to that of Co(II)-carbonic anhydrase. Tentative structures have been proposed for these species.     

Brucella suis carbonic anhydrases and their inhibitors: Towards alternative antibiotics?

Carbonic anhydrases have started to emerge as new potential antibacterial targets for several pathogens. Two β-carbonic anhydrases, denominated bsCA I and bsCA II, have been isolated and characterized from the bacterial pathogen Brucella suis, the causative agent of brucellosis or Malta fever. These enzymes have been investigated in detail and a wide range of classical aromatic and heteroaromatic sulfonamides as well as carbohydrate-based compounds have been found to inhibit selectively and efficiently Brucella suis carbonic anhydrases. Inhibition of these metalloenzymes constitutes a novel approach for the potential development of new anti-Brucella agents. This review aims at discussing the recent literature on this topic.     

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA), an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II) as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO₂ substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.     

Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags

Due to their involvement in diverse pathological conditions, carbonic anhydrases have been the targets of drug developments for the treatments of glaucoma, epilepsy, high altitude sickness, as well as cancer. Of about 14 isozymes of carbonic anhydrases, carbonic anhydrase-II (hCA-II) has been most extensively investigated from the structural, functional, and inhibitor design point of view. We discovered that hCA-II preferentially binds to the Sepharose-iminodiacetate (IDA)-Zn(2+) column, and such binding does not require incorporation of either N- or C-terminal histidine tags in the protein structure. By using the Sepharose-IDA-Zn(2+) affinity column, we purified the Escherichia coli expressed hCA-II with an overall recovery of 76%. The purified enzyme showed a single band on the SDS-PAGE. Due to ease in preparing the Sepharose-IDA-Zn(2+) column, and purifying hCA-II just in one step, the overall protocol will be ideal for producing bulk quantities of the enzyme for high throughput screening of inhibitors.     

Prokaryotic carbonic anhydrases

Carbonic anhydrases catalyze the reversible hydration of CO(2) [CO(2)+H(2)Oright harpoon over left harpoon HCO(3)(-)+H(+)]. Since the discovery of this zinc (Zn) metalloenzyme in erythrocytes over 65 years ago, carbonic anhydrase has not only been found in virtually all mammalian tissues but is also abundant in plants and green unicellular algae. The enzyme is important to many eukaryotic physiological processes such as respiration, CO(2) transport and photosynthesis. Although ubiquitous in highly evolved organisms from the Eukarya domain, the enzyme has received scant attention in prokaryotes from the Bacteria and Archaea domains and has been purified from only five species since it was first identified in Neisseria sicca in 1963. Recent work has shown that carbonic anhydrase is widespread in metabolically diverse species from both the Archaea and Bacteria domains indicating that the enzyme has a more extensive and fundamental role in prokaryotic biology than previously recognized. A remarkable feature of carbonic anhydrase is the existence of three distinct classes (designated alpha, beta and gamma) that have no significant sequence identity and were invented independently. Thus, the carbonic anhydrase classes are excellent examples of convergent evolution of catalytic function. Genes encoding enzymes from all three classes have been identified in the prokaryotes with the beta and gamma classes predominating. All of the mammalian isozymes (including the 10 human isozymes) belong to the alpha class; however, only nine alpha class carbonic anhydrase genes have thus far been found in the Bacteria domain and none in the Archaea domain. The beta class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants as well as enzymes from phylogenetically diverse species from the Archaea and Bacteria domains. The only gamma class carbonic anhydrase that has thus far been isolated and characterized is from the methanoarchaeon Methanosarcina thermophila. Interestingly, many prokaryotes contain carbonic anhydrase genes from more than one class; some even contain genes from all three known classes. In addition, some prokaryotes contain multiple genes encoding carbonic anhydrases from the same class. The presence of multiple carbonic anhydrase genes within a species underscores the importance of this enzyme in prokaryotic physiology; however, the role(s) of this enzyme is still largely unknown. Even though most of the information known about the function(s) of carbonic anhydrase primarily relates to its role in cyanobacterial CO(2) fixation, the prokaryotic enzyme has also been shown to function in cyanate degradation and the survival of intracellular pathogens within their host. Investigations into prokaryotic carbonic anhydrase have already led to the identification of a new class (gamma) and future research will undoubtedly reveal novel functions for carbonic anhydrase in prokaryotes.     

The esterase activity of bovine carbonic anhydrase B above pH 9. Reversible and cooalent inhibition by acetozolamide.

The reversible complex between the metalloenzyme bovine carbonic anhydrase B and the sulfonamide inhibitor acetazolamide can be "frozen" irreversibly by the addition of a covalent bond between the methyl group of the inhibitor and the tau-nitrogen of histidine-64. In both cases the inhibited enzyme is inactive as an esterase toward p-nitrophenyl propionate at physiological pH but retains activity controlled by an ionization in the protein exhibiting a pK-a greater than 10. Similarly, both the covalently and reversibly inhibited enzymes in which the catalytically essential Zn(II) ion has been replaced with Co(II) display the same visible absorption spectrum which is invariant over the pH range from 5 to 12. The evidence therefore indicates that the position of the acetazolamide moiety in the active site is independent of both pH and the presence of the covalent bond to histidine-64. Moreover, when reversibly bound, this inhibitor has been shown to replace the water molecule (or hydroxide ion) known to occupy the fourth coordination position of the metal ion and frequently implicated in the catalytic mechanism of carbonic anhydrases. Thus, the activity exhibited by the inhibited enzymes and consequently the second rise observed in the pH rate profile of the native enzyme above pH 0 cannot reflect the ionization of such a water molecule in contrast to what has been postulated previously (Pocker, Y., and Storm, D. R. (1968) Biochemistry 7, 1202-1214). Displacement of the zinc-bound solvent molecule rather than the alkylation of histidine-64 is suggested, however, as the cause of the inactivation of the alkylated enzyme round neutrality. Taken together, the biphasic pH rate profile of native bovine carbonic anhydrase B as well as the activity retained by the alkylated enzyme above pH 9 are best described by a model in which two groups in the enzyme ionize independently, thereby raising the possibility that the high pH activity is controlled by an ionization outside the active site region of the enzyme. Above pH 9.5 the pK; for the reversible interaction between native carbonic anhydrase and acetazolamide falls off linearly with increasing pH. The slope of --1.56 suggests that, among other factors, more than one ionization is responsible for the descending limb of the pH-i-pH profile.     

The purification and properties of carbonic anhydrases from guinea-pig erythrocytes and the mucosae of the gastrointestinal tract

Procedures for isolating carbonic anhydrase (EC 4.2.1.1) enzymes from the erythrocytes and the mucosae of the gastrointestinal tract of guinea pigs are described. From a haemolysate, haemoglobin was removed by the addition of ammonium sulphate, and also by two other methods, namely by gel filtration or by adsorption on DEAE-Sephadex. The crude enzyme thus obtained was resolved into the different isoenzymes by chromatography with DEAE-cellulose. From particle-free supernatants of homogenates of some gastrointestinal tissues, carbonic anhydrases were purified by ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography with DEAE-cellulose. The major isoenzymes from blood, stomach, proximal colonic mucosa and caecal mucosa were homogeneous during ion-exchange chromatography, acrylamide-gel electrophoresis, and centrifugal examination. From these tissues, carbonic anhydrase was isolated as two major isoenzymes. They resemble the pairs of isoenzymes discovered in the bloods of other species. The carbon dioxide hydratase activity of one isoenzyme (;high activity' carbonic anhydrase) was 40 times that of the other isoenzyme (;low activity' carbonic anhydrase), as measured at a single substrate concentration. Two other minor components of the enzyme are also found in guinea-pig erythrocytes. All of the enzymes isolated had molecular weights of nearly 30000 (sedimentation equilibrium). ;High activity' carbonic anhydrases from blood and gastrointestinal tissues were indistinguishable according to some chemical, physical and kinetic measurements; similarly ;low activity' carbonic anhydrases from those tissues were indistinguishable. ;High activity' carbonic anhydrase was markedly different from the ;low activity' carbonic anhydrase with respect to its amino acid composition, chromatographic behaviour and isoelectric pH value. Marked differences were also found in the tissue concentrations of the major isoenzymes. It is suggested that the characteristic and selective distribution of the different forms of carbonic anhydrase in the guinea-pig tissues is related to the specific and different physiological functions of the enzymes.     

Paramagnetic and fluorescent probes attached to "buried" sulfhydryl groups in human carbonic anhydrases. Application to inhibitor binding, denaturation and refolding.

1. The single -SH groups in the human carbonic anhydrases B and C have been modified under denaturing conditions. The modified enzymes recover catalytic activity after dilution of the denaturing medium with buffer. By this method a spin label and a fluorescent probe were specifically introduced into the molecules. 2. The modified and reactivated enzymes have similar kinetic properties, inhibitor-binding constants, circular dichroism spectra, and stabilities towards guanidine hydrochloride as the native enzymes. However, the esterase activity of the modified C enzyme is reduced to about 50%. 3. The spectra associated with the probes are insensitive to inhibitor binding in case of the B enzyme, whereas changes of electron paramagnetic resonance spectrum and fluorescence intensity respectively, were observed for the probe-containing C enzymes. The cysteines are located in different parts of the tertiary structures of the homologous B and C enzymes, and these observations suggest that small conformational changes accompanying inhibitor binding are localized to regions of the molecules near the active-site cavity. 4. During denaturation of the spin-labeled B enzyme in 1.7 M guanidine hydrochloride a transient mobilization of the probe occurs, but the mobility is ultimately reduced to a low level. This observation supports previous evidence that denaturation under these conditions, in or near the transition region, mainly yields incorrectly folded molecules rather than stable intermediates between native and fully denatured molecules. 5. During refolding of fully denatured, spin-labeled B and C enzymes the mobility of the probe is drastically reduced within less than 0.1 s after dilution. This would reflect a very short lifetime of the randomly coiled state under these conditions.     

Inhibition of the β-carbonic anhydrases from Mycobacterium tuberculosis with C-cinnamoyl glycosides: Identification of the first inhibitor with anti-mycobacterial activity

A small series of C-cinnamoyl glycoside containing the phenol moiety was tested for the inhibition of the three Mycobacterium tuberculosis β-carbonic anhydrases (CAs, EC 4.2.1.1) with activities in the low micromolar range detected. The compounds were also tested for the inhibition of growth of M. tuberculosis H₃₇Rv strain, leading to the identification of (E)-1-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosyl)-4-(3-hydroxyphenyl)but-3-en-2-one (1) as the first carbonic anhydrase inhibitor with anti-tubercular activity.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

Rat renal and erythrocyte carbonic anhydrases. Purification and properties.

Rat renal and erythrocyte carbonic anhydrases (carbonate hydro-lyase, EC 4.2.1.1) were isolated by affinity chromatography. The erythrocytes contain two major forms of the enzyme. One of the forms has a specific activity (towards CO2) 30 times higher than the other and constitutes the major part of the total cellular carbonic anhydrase. The amino acid compositions of this high-activity type and of the low-activity type are similar to the compositions reported for these types in other species. The kidney appears to have only one high-activity form of carbonic anhydrase which is very similar to and probably identical with the erythrocyte high-activity form.     

A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila

The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-). Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site. On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.     

Carbonic anhydrase in Escherichia coli. A product of the cyn operon.

The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases. but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate.