The sequestration of hydroxyl ions by CO2 in liquid water: the physiological implications and the second function of carbonic anhydrase

The pH changes due to bubbling CO2 through water produced anomalies which were more readily explained by an hypothesis based on electrostatic attractions between the molecules. The present studies have suggested that an hexagonal array of six carbon dioxide molecules could bind and sequester a hydroxyl anion. The binding energy of the complex is estimated to be comparable with that of a covalent compound and its dissociation may only occur at the water interface with air or at the water/hydrophobic protein interface in a protein cleft. The physiological importance lies in the consequential release of an equal number of free hydrogen ions (H3O+) and the disruption of the normal action of buffer systems in regulating the cytoplasmic pH. The counteraction of this sequestration reaction and the acid-base disturbances which result, form the second important function of carbonic anhydrase isoforms, the mechanisms of which are briefly discussed.     

Structure,function and applications of carbonic anhydrase isozymes

The carbonic anhydrases enzymes (CAs, EC 4.2.1.1) are zinc containing metalloproteins, which efficiently catalyse the reversible conversion of carbon dioxide to bicarbonate and release proton. These enzymes are essentially important for biological system and play several important physiological and patho-physiological functions. There are 16 different alpha-carbonic anhydrase isoforms studied, differing widely in their cellular localization and biophysical properties. The catalytic domains of all CAs possess a conserved tertiary structure fold, with predominately β-strands. We performed an extensive analysis of all 16 mammalian CAs for its structure and function in order to establish a structure–function relationship. CAs have been a potential therapeutic target for many diseases. Sulfonamides are considered as a strong and specific inhibitor of CA, and are being used as diuretics, anti-glaucoma, anti-epileptic, anti-ulcer agents. Currently CA inhibitors are widely used as a drug for the treatment of neurological disorders, anti-glaucoma drugs, anti-cancer, or anti-obesity agents. Here we tried to emphasize how CAs can be used for drug discovery, design and screening. Furthermore, we discussed the role of CA in carbon capture, carbon sensor and metabolon. We hope this review provide many useful information on structure, function, mechanism, and applications of CAs in various discipline.     

Carbonic anhydrase inhibitors. Inhibition of human tumor-associated isozymes IX and cytosolic isozymes I and II with some 1,3,4-oxadiazole-thiols

A series of chiral 1,3,4-oxadiazole-5-thiols incorporating 2-substituted-benzenesulfonamide moieties has been prepared from amino acids, via the ester and carbohydrazide intermediate, followed by cyclization with carbon disulfide. Some of these compounds have been investigated for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the human cytosolic hCA I and II, and the human, transmembrane, tumor-associated isozyme hCA IX. All these compounds showed weak (millimolar) affinity for the three isozymes, except two carbohydrazides and two heterocyclic thiols which selectively inhibited the tumor-associated isozyme with inhibition constants around 10 microM. Such compounds constitute interesting lead molecules for the possible design of CA IX-selective inhibitors.     

Characterization of two novel nodule-enhanced α-type carbonic anhydrases from Lotus japonicus

Two cDNA clones coding for α-type carbonic anhydrases (CA; EC 4.2.1.1) in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the full-length proteins was confirmed by heterologous expression in Escherichia coli and purification of the encoded polypeptides. The developmental expression pattern of LjCAA1 and LjCAA2 revealed that both genes code for nodule enhanced carbonic anhydrase isoforms, which are induced early during nodule development. The genes were slightly to moderately down-regulated in ineffective nodules formed by mutant Mesorhizobium loti strains, indicating that these genes may also be involved in biochemical and physiological processes not directly linked to nitrogen fixation/assimilation. The spatial expression profiling revealed that both genes were expressed in nodule inner cortical cells, vascular bundles and central tissue. These results are discussed in the context of the possible roles of CA in nodule carbon dioxide (CO(2)) metabolism.     

Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase activity

Abstract

Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase’s. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases.     

A comparative study of carbonic anhydrase activity in lymphocytes from colorectal cancer tissues and adjacent healthy counterparts

Several carbonic anhydrase (CA, EC 4.2.1.1) isoforms play an essential role in processes connected to tumorigenesis, as they efficiently accelerate the hydration of carbon dioxide to bicarbonate and proton. In this context, examples are CA IX and CA XII, which were proved to be upregulated in many solid malignancies. On the other hand, cancer and the immune system are inextricably linked, and targeting the immune checkpoints recently was shown to efficiently improve the treatment of malignancies. In this study, we have investigated the expression of CA isoforms in tumour-infiltrating lymphocytes (TILs) that, according to the immunosurveillance theory, were suggested to have a crucial role in the development of colorectal cancer (CRC). T lymphocytes isolated from healthy surrounding mucosa showed a higher CA activity compared to those present in tumour and peripheral blood in the same patients. CA I and II were confirmed as enzyme isoforms involved in the process, as determined by proteomic analysis of corresponding TIL samples. These preliminary findings suggest a dysregulation of the local immune response in the CRC tissues and a loss of effective anticancer mechanisms mediated by CAs therein.     

A search for the function of human carbonic anhydrase B.

Because of the very high activity and abundance of human red cell carbonic anhydrase C (carbamate hydrolase, EC 4.2.1.1), it seemed likely that the second isozyme, B, might not be essential for CO2 metabolism. It was then found that physiological concentrations of Cl- inhibited catalysis of CO2 hydration by the B enzyme (but not by type C), suggesting further that type B does not function in vivo as a carbonic anhydrase. The versatility of the catalytic activity of carbonic anhydrase for a number of 'artificial' substrates suggested that enzyme B may be utilized in reactions of intermediary metabolism. A number of hydration, dehydration, decarboxylation, kinase, and phosphatase systems were tested to determine a possible physiological function for the enzyme. Results with eighteen possible substrates were negative and the possibility is discussed that mammalian carbonic anhydrase B is an evolutionary accident.     

Molecular modeling study for the binding of zonisamide and topiramate to the human mitochondrial carbonic anhydrase isoform VA

Zonisamide and topiramate are two antiepileptic drugs known to induce weight loss in epilepsy patients. These molecules were recently shown to act as carbonic anhydrase (CA) inhibitors, being presumed that the weight loss may be due to the inhibition of the mitochondrial isozymes CA VA and CA VB involved in metabolic processes, among which lipid biosynthesis. To better understand the interaction of these compounds with CAs, here, we report a homology modeling and molecular dynamics simulations study on their adducts with human carbonic anhydrase VA (hCA VA). According to our results, in both cases the inhibitor sulfamate/sulfonamide moiety participates in the canonical interactions with the catalytic zinc ion, whereas the organic scaffold establishes a large number of van der Waals and polar interactions with the active site cleft. A structural comparison of these complexes with the corresponding homologues with human carbonic anhydrase II (hCA II) provides a rationale to the different affinities measured for these drugs toward hCA VA and hCA II. In particular, our data suggest that a narrower active site cleft, together with a different hydrogen bond network arrangement of hCA VA compared to hCA II, may account for the different Kd values of zonisamide and topiramate toward these physiologically relevant hCA isoforms. These results provide useful insights for future design of more isozyme-selective hCA inhibitors with potential use as anti-obesity drugs possessing a novel mechanism of action.     

Carbonic anhydrase inhibitors: copper(II) complexes of polyamino-polycarboxylamido aromatic/heterocyclic sulfonamides are very potent inhibitors of the tumor-associated isoforms IX and XII

Reaction of EDTA/DTPA dianhydride with aromatic/heterocyclic sulfonamides afforded a series of derivatives incorporating polyaminopolycarboxylate tails and benzenesulfonamide or 1,3,4-thiadiazole-2-sulfonamide heads. These compounds have been used as ligands to prepare Cu(II) complexes. Both parent sulfonamides as well as their copper complexes behaved as potent inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, and transmembrane CA IX and XII. Some Cu(II) complexes showed subnanomolar affinities and some selectivity for the inhibition of the tumor-associated isoforms IX and XII and might be used as PET hypoxia markers of tumors.     

Nucleotide sequence and structure of the mouse carbonic anhydrase III gene

At least 14 distinct isozymes of carbonic anhydrase have been identified in mammals. These enzymes catalyze the hydration of carbon dioxide and are essential for regulation of cellular pH and carbon dioxide transport. Carbonic anhydrase III is highly expressed in certain tissues, including muscle and fat where it constitutes up to 25% of the soluble protein. We cloned a cDNA encoding mouse carbonic anhydrase III. This cDNA contains 1653 bp, consisting of 79 bp in the 5' UTR, a 780 bp open reading frame, and 794 bp of the 3' UTR, including two potential polyadenylation signals. Fluorescent in situ hybridization confirmed the existence of a single copy of the gene on chromosome 3. We then isolated the genomic DNA for mouse carbonic anhydrase III and analyzed its structure. The gene consists of seven exons and six introns which span 10.5 kb. The 5' flanking region of the genomic DNA is notable for a pyrimidine rich region consisting of two dinucleotide repeats containing 23 and 20 TC pairs separated by the same 15 bp spacer.     

碳酸酐酶在聚甲基戊烯中空纤维膜表面的固定及其性能分析

采用两步法将碳酸酐酶共价键合在聚甲基戊烯(Polymethyl-pentene,PMP)膜式氧合器表面以提高其清除血液中CO2的能力。首先采用等离子体处理法将羟基引入PMP表面,然后用偶联剂溴化氰(CNBr)将碳酸酐酶(Carbonic anhydrase,CA)固定在PMP膜表面。采用XPS、表面接触角测定仪对等离子体处理后材料表面的物理化学性质进行了表征。以对硝基苯酚乙酸酯(p-nitrophenyl acetate,p-NPA)为底物,采用紫外分光光度计测定了接枝CA的活性、浓度、重复利用性、储存稳定性。结果表明,等离子体处理方法能将羟基成功引入PMP表面;CA能被成功地偶联在无活性官能团聚合物表面,在保持酶活性的同时获得较高的接枝效率;共价接枝CA(Covalently immobilized CA,CACI)的浓度随CNBr浓度的增加而增加,最大可达理论单分子层接枝量的73%;CACI比物理吸附的CA(Physically adsorbed CA,CAPA)具更好的重复利用性;37oC下,CACI比CA溶液表现出更好的储存稳定性。本方法有望应用在膜式氧合器上以提高其对血液中CO2的排除效率。     

Carbon concentration mechanisms in photosynthetic microorganisms

Unicellular green algae and cyanobacteria have mechanism to actively concentrate dissolved inorganic carbon into the cells, only if they are grown with air levels of CO2. The carbon concentration mechanisms are commonly known as "CCM" or "DIC-pumps". The DIC-pumps are environmental adaptation that function to actively transport and accumulate inorganic carbon (HCO3- and CO2; Ci) within the cell and then uses this Ci pool to actively increase the concentration of CO2 at the site of ribulose bisphosphate carboxylase-oxygenase (Rubisco), the primary CO2-fixing enzyme. The current working model for dissolved inorganic carbon concentration mechanism in unicellular green algae includes several isoforms of carbonic anhydrase (CA), and ATPase driven active transporters at the plasmalemma and at the inner chloroplast envelopes. In the past fifteen years, significant progress has been made in isolating and characterizing the various isoforms of carbonic anhydrase at the biochemical and molecular level. However, we have an inadequate understanding of active transporters that are located on the plasmalemma and at the chloroplast envelopes. In this mini-review we focus on certain aspects of the induction, function and significance of the dissolved inorganic carbon concentration mechanisms in aquatic photosynthetic microorganisms.     

Carbon Dioxide Hydration with Liposomes Entrapping Carbonic Anhydrase

Abstract

Liposomes entrapping enzymes behave as small membrane bioreactors which can be used in biotechnological and biomedical applications. Furthermore, liposomes loading enzymes are being used as model to mimic cellular and subcellular organel behaviour. This work deals with the study of the kinetic behaviour of carbonic anhydrase (CA) entrapped in lipid vesicle. CA is nearly ubiquitous, being present in many tissues of different species, in cells, subcellular particles and membranes. The carbonic anhydrase entrapment in liposomes, as a function of the lipid cocktail used, was studied preliminarily. Then kinetics of carbon dioxide hydration reaction, catalysed by CA entrapped in liposomes, has been studied.     

Potent isothiocyanate inhibitors of carbonic anhydrase: synthesis and evaluation

The reversible hydration of carbon dioxide by carbonic anhydrase (CA) regulates pH and carbon dioxide concentrations in diverse biological systems. Potent irreversible inhibition of CA would facilitate study of the dynamics of CA turnover as well as therapeutic effects due to long-term inhibition of the enzyme. We have synthesized isothiocyanate-containing sulfonamide inhibitors of CA from the corresponding aminosulfonamides. Significant increases in apparent binding of some of the isothiocyanate inhibitors over the amine analogues were consistent with covalent inhibition of the enzyme.     

Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.     

Differential in vitro inhibition effects of some antibiotics on tumor associated carbonic anhydrase isozymes of hCA-IX and hCA-XII

Hypoxia is a common characteristic of locally advanced solid tumors that has been associated with diminished therapeutic response and malignant progression. Human carbonic anhydrase (hCA) hCA IX and XII isozymes are tumor associated isoforms which contribute to acidification of the tumor environment by catalyzing the hydration of carbon dioxide to bicarbonate and protons.In the present study our goal was to investigate the inhibition effects of 15 different antibiotics belonging to the following classes: Lactams, cephalosporins, macrolides etc., on the tumor associated carbonic anhydrase isozymes hCA-IX, hCA-XII and cytosolic carbonic anhydrase hCA-I and hCA-II.     

Differential in vitro inhibition effects of some antibiotics on tumor associated carbonic anhydrase isozymes of hCA-IX and hCA-XII

Hypoxia is a common characteristic of locally advanced solid tumors that has been associated with diminished therapeutic response and malignant progression. Human carbonic anhydrase (hCA) hCA IX and XII isozymes are tumor associated isoforms which contribute to acidification of the tumor environment by catalyzing the hydration of carbon dioxide to bicarbonate and protons.In the present study our goal was to investigate the inhibition effects of 15 different antibiotics belonging to the following classes: Lactams, cephalosporins, macrolides etc., on the tumor associated carbonic anhydrase isozymes hCA-IX, hCA-XII and cytosolic carbonic anhydrase hCA-I and hCA-II.     

Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase

Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 μM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.     

A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains

This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (Ci) from the surrounding seawater to the symbiotic algae that are found within the clam's tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This alpha-family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N-terminal CA domain and a C-terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and contains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached/secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it contains a dual domain structure unique amongst all animal CAs characterized to date.