Publications

• Carbonic anhydrase inhibitors.

  Supuran C.T., Scozzafava A., Casini A.

  At least 14 different carbonic anhydrase (CA, EC 4.2.1.1) isoforms were isolated in higher vertebrates, where these zinc enzymes play crucial physiological roles. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII), others are membrane-bound (CA IV, CA IX, CA XII, and CA XIV), CA V ... >> More

  At least 14 different carbonic anhydrase (CA, EC 4.2.1.1) isoforms were isolated in higher vertebrates, where these zinc enzymes play crucial physiological roles. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII), others are membrane-bound (CA IV, CA IX, CA XII, and CA XIV), CA V is mitochondrial and CA VI is secreted in saliva. Three acatalytic forms are also known, which are denominated CA related proteins (CARP), CARP VIII, CARP X, and CARP XI. Several important physiological and physio-pathological functions are played by many CA isozymes, which are strongly inhibited by aromatic and heterocyclic sulfonamides as well as inorganic, metal complexing anions. The catalytic and inhibition mechanisms of these enzymes are understood in detail, and this helped the design of potent inhibitors, some of which possess important clinical applications. The use of such enzyme inhibitors as antiglaucoma drugs will be discussed in detail, together with the recent developments that led to isozyme-specific and organ-selective inhibitors. A recent discovery is connected with the involvement of CAs and their sulfonamide inhibitors in cancer: several potent sulfonamide inhibitors inhibited the growth of a multitude of tumor cells in vitro and in vivo, thus constituting interesting leads for developing novel antitumor therapies. Furthermore, some other classes of compounds that interact with CAs have recently been discovered, some of which possess modified sulfonamide or hydroxamate moieties. Some sulfonamides have also applications as diagnostic tools, in PET and MRI or as antiepileptics or for the treatment of other neurological disorders. Future prospects for drug design applications for inhibitors of these ubiquitous enzymes are also discussed. << Less

  Med Res Rev 23:146-189(2003) [PubMed] [EuropePMC]

• Prokaryotic carbonic anhydrases.

  Smith K.S., Ferry J.G.

  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 al ... >> More

  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. << Less

  FEMS Microbiol Rev 24:335-366(2000) [PubMed] [EuropePMC]

• Refined structure of human carbonic anhydrase II at 2.0-A resolution.

  Eriksson A.E., Jones T.A., Liljas A.

  The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure-factor least-squares refinement at 2.0 A resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stab ... >> More

  The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure-factor least-squares refinement at 2.0 A resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor-acceptor relations in the active site can be assigned and are described here in detail. The O gamma 1 atom of Thr-199 donates its proton to the O epsilon 1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds. << Less

  Proteins 4:274-282(1988) [PubMed] [EuropePMC]

• Tobacco nectarin III is a bifunctional enzyme with monodehydroascorbate reductase and carbonic anhydrase activities.

  Carter C.J., Thornburg R.W.

  Tobacco plants secrete a limited array of proteins (nectarins) into their floral nectar. N-terminal sequencing of the Nectarin II ( NEC2; 35kD) and the Nectarin III ( NEC3; 40kD) proteins revealed that they both share identity with dioscorin, the major soluble protein of yam tubers. These sequence ... >> More

  Tobacco plants secrete a limited array of proteins (nectarins) into their floral nectar. N-terminal sequencing of the Nectarin II ( NEC2; 35kD) and the Nectarin III ( NEC3; 40kD) proteins revealed that they both share identity with dioscorin, the major soluble protein of yam tubers. These sequences also revealed that NEC2 is a breakdown product of NEC3. Using these N-terminal peptide sequences, degenerate oligonucleotides were designed that permitted the isolation of a partial NEC3 cDNA. This cDNA was then used to probe a nectary specific cDNA library and a full-length NEC3 cDNA clone was isolated. Complete sequence analysis confirmed the identity of NEC3 as a dioscorin-like protein. MALDI-TOF mass spectrometric fingerprinting of tryptic peptides derived from the purified NEC3 confirmed that this protein was encoded by the isolated cDNA. NEC3 was shown to possess both carbonic anhydrase and monodehydroascorbate reductase activities. RT-PCR based expression analyses demonstrated that NEC3 transcript is expressed throughout nectary development as well as in other floral organs. A proposed function in the maintenance of pH and oxidative balance in nectar is discussed. << Less

  Plant Mol. Biol. 54:415-425(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure and mechanism of carbonic anhydrase.

  Lindskog S.

  Carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) is a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide: CO2+ H2O<-->HCO3(-)+H+. The enzyme is the target for drugs, such as acetazolamide, methazolamide, and dichlorphenamide, for the treatment of glaucoma. T ... >> More

  Carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) is a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide: CO2+ H2O<-->HCO3(-)+H+. The enzyme is the target for drugs, such as acetazolamide, methazolamide, and dichlorphenamide, for the treatment of glaucoma. There are three evolutionarily unrelated CA families, designated alpha, beta, and gamma. All known CAs from the animal kingdom are of the alpha type. There are seven mammalian CA isozymes with different tissue distributions and intracellular locations, CA I-VII. Crystal structures of human CA I and II, bovine CA III, and murine CA V have been determined. All of them have the same tertiary fold, with a central 10-stranded beta-sheet as the dominating secondary structure element. The zinc ion is located in a cone-shaped cavity and coordinated to three histidyl residues and a solvent molecule. Inhibitors bind at or near the metal center guided by a hydrogen-bonded system comprising Glu-106 and Thr-199. The catalytic mechanism of CA II has been studied in particular detail. It involves an attack of zinc-bound OH-on a CO2 molecule loosely bound in a hydrophobic pocket. The resulting zinc-coordinated HCO3-ion is displaced from the metal ion by H2O. The rate-limiting step is an intramolecular proton transfer from the zinc-bound water molecule to His-64, which serves as a proton shuttle between the metal center and buffer molecules in the reaction medium. << Less

  Pharmacol Ther 74:1-20(1997) [PubMed] [EuropePMC]

• Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana.

  Buren S., Ortega-Villasante C., Blanco-Rivero A., Martinez-Bernardini A., Shutova T., Shevela D., Messinger J., Bako L., Villarejo A., Samuelsson G.

  <h4>Background</h4>The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harb ... >> More

  <h4>Background</h4>The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.<h4>Methodology/principal findings</h4>Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.<h4>Conclusions/significance</h4>We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast. << Less

  PLoS ONE 6:E21021-E21021(2011) [PubMed] [EuropePMC]

• Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide.

  Huang S., Xue Y., Sauer-Eriksson E., Chirica L., Lindskog S., Jonsson B.-H.

  The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group ... >> More

  The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II. << Less

  J. Mol. Biol. 283:301-310(1998) [PubMed] [EuropePMC]

• Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant.

  Tu C.K., Silverman D.N., Forsman C., Jonsson B.H., Lindskog S.

  To test the hypothesis that histidine 64 in the active site of human carbonic anhydrase II functions as a proton-transfer group in the catalysis of CO2 hydration, we have studied a site-specific mutant having histidine 64 replaced by alanine, which cannot transfer protons. The steady-state kinetic ... >> More

  To test the hypothesis that histidine 64 in the active site of human carbonic anhydrase II functions as a proton-transfer group in the catalysis of CO2 hydration, we have studied a site-specific mutant having histidine 64 replaced by alanine, which cannot transfer protons. The steady-state kinetics of CO2 hydration has been measured as well as the exchange of 18O between CO2 and water at chemical equilibrium. The results show that the rate of exchange between CO2 and HCO3-at chemical equilibrium is essentially unaffected by the amino acid substitution at pH greater than 7.0 and slightly decreased in the mutant at pH less than 7.0 (by a factor of 2 at pH 6.0). However, in the absence of buffer the rate of release from the active site of water bearing substrate oxygen is smaller by as much as 20-fold for the mutant as compared to unmodified enzyme. Furthermore, in the unmodified enzyme water release is inhibited by micromolar concentrations of Cu2+ ions, but no such inhibition is observed with the alanine 64 variant. These results suggest that the mutation has specifically affected the rate of proton transfer between the active site and the reaction medium. This kinetic defect in the mutant can be overcome by increasing the concentration of certain buffers, such as imidazole and 1-methylimidazole, but not by others buffers, such as MOPS or HEPES. Similarly, the maximal rate of CO2 hydration at steady state catalyzed by the alanine 64 variant is very low in the presence of MOPS or TAPS buffers but considerably higher in the presence of imidazole derivatives.(ABSTRACT TRUNCATED AT 250 WORDS) << Less

  Biochemistry 28:7913-7918(1989) [PubMed] [EuropePMC]

• Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II.

  Fisher Z., Hernandez Prada J.A., Tu C., Duda D., Yoshioka C., An H., Govindasamy L., Silverman D.N., McKenna R.

  In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine an ... >> More

  In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH on proton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and two double mutants: H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chains extending into the active-site cavity with distances from the zinc approximately equivalent to that of His64. Crystal structures were determined at pH 5.1-10.0, and the catalysis of the exchange of (18)O between CO(2) and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 10(5) s(-)(1) was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at a rate near 10(3) s(-)(1) was observed for His62. The observation, in the crystal structures, of a completed hydrogen-bonded water chain between the histidine shuttle residue and the zinc-bound solvent does not appear to be required for efficient proton transfer. The data suggest that the number of intervening water molecules between the donor and acceptor supporting efficient proton transfer in HCA II is important, and furthermore suggest that a water bridge consisting of two intervening water molecules is consistent with efficient proton transfer. << Less

  Biochemistry 44:1097-1105(2005) [PubMed] [EuropePMC]