Carbonic Anhydrases and Their Biotechnological Applications
Abstract
:1. Introduction
2. Artificial Lungs
3. Biosensors
4. CO2 Sequestration
5. Pharmalogical Considerations
6. Blood Substitutes
7. Conclusions
Conflict of Interest
References
- Aggarwal, M.; Boone, C.D.; Kondeti, B.; McKenna, R. Structural annotation of human carbonic anhydrases. J. Enzyme Inhib. Med. Chem. 2013, 28, 267–277. [Google Scholar] [CrossRef]
- Krishnamurthy, V.M.; Kaufman, G.K.; Urbach, A.R.; Gitlin, I.; Gudiksen, K.L.; Weibel, D.B.; Whitesides, G.M. Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding. Chem. Rev. 2008, 108, 946–1051. [Google Scholar] [CrossRef]
- Rowlett, R.S. Structure and catalytic mechanism of the beta-carbonic anhydrases. Biochim. et Biophy. Acta 2010, 1804, 362–373. [Google Scholar]
- Supuran, C.T. Carbonic anhydrases—an overview. Curr. Pharm. Des. 2008, 14, 603–614. [Google Scholar] [CrossRef]
- Hewett-Emmett, D.; Tashian, R.E. Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families. Mol. Phylogenetics Evolut. 1996, 5, 50–77. [Google Scholar] [CrossRef]
- Lindskog, S. Structure and mechanism of carbonic anhydrase. Pharmacol. Ther. 1997, 74, 1–20. [Google Scholar] [CrossRef]
- Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C.T.; de Simone, G. Multiple binding modes of inhibitors to carbonic anhydrases: How to design specific drugs targeting 15 different isoforms? Chem. Rev. 2012, 112, 4421–4468. [Google Scholar] [CrossRef]
- Bergenhem, N.C.; Hallberg, M.; Wisén, S. Molecular characterization of the human carbonic anhydrase-related protein (hca-rp viii). Biochim. et Biophy. Acta 1998, 1384, 294–298. [Google Scholar]
- Sly, W.S.; Hu, P.Y. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu. Rev. Biochem. 1995, 64, 375–401. [Google Scholar] [CrossRef]
- Supuran, C.T.; Scozzafava, A. Carbonic anhydrases as targets for medicinal chemistry. Bioorganic Med. Chem. 2007, 15, 4336–4350. [Google Scholar] [CrossRef]
- Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C.T. Carbonic anhydrases: Current state of the art, therapeutic applications and future prospects. J. Enzyme Inhib. Med. Chem. 2004, 19, 199–229. [Google Scholar] [CrossRef]
- Aggarwal, M.; McKenna, R. Update on carbonic anhydrase inhibitors: A patent review (2008–2011). Expert Opin. Ther. Pat. 2012, 22, 903–915. [Google Scholar]
- Chegwidden, W.R.; Carter, N.D. Introduction to the Carbonic Anhydrases. In The Carbonic Anhdyrases: New horizons; Chegwidden, W.R., Carter, N.D., Edwards, Y.H., Eds.; Birkhäuser Verlag: Boston, MA, USA, 2000; pp. 13–29. [Google Scholar]
- Christianson, D.W.; Fierke, C.A. Carbonic anhydrase: Evolution of the zinc binding site by nature and design. Acc. Chem. Res. 1996, 29, 331–339. [Google Scholar] [CrossRef]
- Duda, D.; McKenna, R. Carbonic Anhydrase, α-class. In Handbook of Metalloproteins; Messerschmidt, A., Ed.; John Wiley & Sons: New York, NY, USA, 2004; pp. 249–263. [Google Scholar]
- Lindskog, S.; Coleman, J.E. Catalytic mechanism of carbonic-anhydrase. Proc. Natl. Acad. Sci. USA 1973, 70, 2505–2508. [Google Scholar] [CrossRef]
- Lindskog, S.; Silverman, D.N. The Catalytic Mechanism of Mammalian Carbonic Anhydrases. In The Carbonic Anhdyrases: New Horizons; Chegwidden, W.R., Carter, N.D., Edwards, Y.H., Eds.; Birkhäuser Verlag: Boston, MA, USA, 2000; pp. 175–195. [Google Scholar]
- Tu, C.K.; Silverman, D.N.; Forsman, C.; Jonsson, B.H.; Lindskog, S. Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase ii studied with a site-specific mutant. Biochemistry 1989, 28, 7913–7918. [Google Scholar] [CrossRef]
- Mikulski, R.L.; Silverman, D.N. Proton transfer in catalysis and the role of proton shuttles in carbonic anhydrase. Biochim. et Biophy. Acta 2010, 1804, 422–426. [Google Scholar] [CrossRef]
- Silverman, D.N. Carbonic anhydrase: Oxygen-18 exchange catalyzed by an enzyme with rate-contributing proton-transfer steps. Methods Enzymol. 1982, 87, 732–752. [Google Scholar] [CrossRef]
- Silverman, D.N.; Lindskog, S. The catalytic mechanism of carbonic anhydrase: Implications of a rate-limiting protolysis of water. Acc. Chem. Res. 1988, 21, 30–36. [Google Scholar] [CrossRef]
- Silverman, D.N.; McKenna, R. Solvent-mediated proton transfer in catalysis by carbonic anhydrase. Acc. Chem. Res. 2007, 40, 669–675. [Google Scholar] [CrossRef]
- Avvaru, B.S.; Busby, S.A.; Chalmers, M.J.; Griffin, P.R.; Venkatakrishnan, B.; Agbandje-McKenna, M.; Silverman, D.N.; McKenna, R. Apo-human carbonic anhdrase ii revisited: Implications of the loss of a metal in protein structure, stability, and solvent network. Biochemistry 2009, 48, 7365–7372. [Google Scholar] [CrossRef]
- Murakami, H.; Marelich, G.P.; Grubb, J.H.; Kyle, J.W.; Sly, W.S. Cloning, expression, and sequence homologies of cdna for human carbonic anhydrase ii. Genomics 1987, 1, 159–166. [Google Scholar] [CrossRef]
- Krebs, J.F.; Fierke, C.A. Determinants of catalytic activity and stability of carbonic anhydrase ii as revealed by random mutagenesis. J. Biol. Chem. 1993, 268, 27458–27466. [Google Scholar]
- Osborne, W.R.; Tashian, R.E. An improved method for the purification of carbonic anhydrase isozymes by affinity chromatography. Anal. Biochem. 1975, 64, 297–303. [Google Scholar] [CrossRef]
- Avvaru, B.S.; Kim, C.U.; Sippel, K.H.; Gruner, S.M.; Agbandje-McKenna, M.; Silverman, D.N.; McKenna, R. A short, strong hydrogen bond in the active site of human carbonic anhydrase ii. Biochemistry 2010, 49, 249–251. [Google Scholar] [CrossRef]
- Eriksson, A.E.; Jones, T.A.; Liljas, A. Refined structure of human carbonic anhydrase ii at 2.0 a resolution. Proteins 1988, 4, 274–282. [Google Scholar] [CrossRef]
- Fisher, S.Z.; Aggarwal, M.; Kovalesky, A.; Silverman, D.N.; McKenna, R. Neutron-diffraction of acetazolamide-bound human carbonic anhydrase ii reveals atomic details of drug binding. J. Am. Chem. Soc. 2012, 134, 14726–14729. [Google Scholar] [CrossRef]
- Fisher, S.Z.; Kovalevsky, A.Y.; Domsic, J.F.; Mustyakimov, M.; McKenna, R.; Silverman, D.N.; Langan, P.A. Neutron structure of human carbonic anhydrase ii: Implications for proton transfer. Biochemistry 2010, 49, 415–421. [Google Scholar] [CrossRef]
- Fisher, S.Z.; Tu, C.; Bhatt, D.; Govindasamy, L.; Agbandje-McKenna, M.; McKenna, R.; Silverman, D.N. Speeding up proton transfer in a fast enzyme: Kinetic and crystallographic studies on the effect of hydrophobic amino acid substitutions in the active site of human carbonic anhydrase ii. Biochemistry 2007, 46, 3803–3813. [Google Scholar] [CrossRef]
- Mikulski, R.; West, D.; Sippel, K.H.; Avvaru, B.S.; Aggarwal, M.; Tu, C.; McKenna, R.; Silverman, D.N. Water networks in fast proton transfer during catalysis by human carbonic anhydrase ii. Biochemistry 2013, 52, 125–131. [Google Scholar] [CrossRef]
- Fisher, Z.; Boone, C.D.; Biswas, S.M.; Venkatakrishnan, B.; Aggarwal, M.; Tu, C.; Agbandje-McKenna, M.; Silverman, D.; McKenna, R. Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase ii. Protein Eng. Des. Sel. PEDS 2012, 25, 347–355. [Google Scholar] [CrossRef]
- Mårtensson, L.-G.; Karlsson, M.; Carlsson, U. Dramatic stabilization of the native state of human carbonic anhydrase ii by an engineered disulfide bond. Biochemistry 2002, 41, 15867–15875. [Google Scholar] [CrossRef]
- Boone, C.D.; Habibzadegan, A.; Tu, C.; Silverman, D.N.; McKenna, R. Structural and catalytic characterization of a thermally stable and acid-stable variant of human carbonic anhydrase ii containing an engineered disulfide bond. Acta Crystallogr. Sect. D Biol. Crystallogr. 2013, 69, 1414–1422. [Google Scholar] [CrossRef]
- Boone, C.D.; Gill, S.; Habibzadegan, A.; McKenna, R. Carbonic anhydrases and their industrial applications. Curr. Top. Biochem. Res. 2013, 14, 1–10. [Google Scholar]
- Christianson, D.W.; Fierke, C.A. Carbonic anhydrase: Evolution of the zinc binding site by nature and by design. Acc. Chem. Res. 1996, 29, 331–339. [Google Scholar] [CrossRef]
- Supuran, C.T. Carbonic anhydrase inhibitors. Bioorganic Med. Chem. Lett. 2010, 20, 3467–3474. [Google Scholar] [CrossRef]
- Supuran, C.T. Inhibition of carbonic anhydrase ix as a novel anticancer mechanism. World J. Clin. Oncol. 2012, 3, 98–103. [Google Scholar] [CrossRef]
- Supuran, C.T. Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nat. Rev. Drug Discov. 2008, 7, 168–181. [Google Scholar] [CrossRef]
- Ware, L.B.; Matthay, M.A. The acute respiratory distress syndrome. N. Engl. J. Med. 2000, 342, 1334–1349. [Google Scholar] [CrossRef]
- Esteban, A.; Anzueto, A.; Frutos, F.; Alia, I.; Brochard, L.; Stewart, T.E.; Benito, S.; Epstein, S.K.; Apezteguia, C.; Nightingale, P.; et al. Characteristics and outcomes in adult patients receiving mechanical ventilation: A 28-day international study. JAMA 2002, 287, 345–355. [Google Scholar] [CrossRef]
- Maggiore, S.M.; Richard, J.C.; Brochard, L. What has been learnt from p/v curves in patients with acute lung injury/acute respiratory distress syndrome. Eur. Respir. J. 2003, 22, 22s–26s. [Google Scholar] [CrossRef]
- Haft, J.W.; Griffith, B.P.; Hirschl, R.B.; Bartlett, R.H. Results of an artificial-lung survey to lung transplant program directors. J. Heart Lung Transplant. 2002, 21, 467–473. [Google Scholar] [CrossRef]
- Hattler, B.G.; Federspiel, W.J. Gas Exchange in the Venous System: Support for the Failing Lung. In The Artificial Lung; Vaslef, S.N., Anderson, R.W., Eds.; Landes Bioscience: Georgetown, DC, USA, 2002; pp. 133–174. [Google Scholar]
- Wegner, J.A. Oxygenator anatomy and function. J. Cardiothorac. Vasc. Anesth. 1997, 11, 275–281. [Google Scholar] [CrossRef]
- Federspiel, W.J.; Henchir, K.A. Artificial Lungs: Basic Principles and Current Applications. In Encyclopedia of Biomaterials and Biomedical Engineering; Wnek, G.E., Bowlin, G.L., Eds.; Marcel Dekker, Inc: New York, NY, USA, 2004; pp. 922–931. [Google Scholar]
- Beckley, P.D.; Holt, D.W.; Tallman, R.D. Oxygenators for Extracorporeal Circulation. In Cardiopulmonary Bypass: Principles and Techniques of Extracorporeal Circulation; Mora, C.T., Ed.; Springer-Verlag: New York, NY, USA, 1995; pp. 199–219. [Google Scholar]
- Okamoto, T.; Tashiro, M.; Sakanashi, Y.; Tanimoto, H.; Imaizumi, T.; Sugita, M.; Terasaki, H. A new heparin-bonded dense membrane lung combined with minimal systemic heparinization prolonged extracorporeal lung assist in goats. Artif. Organs 1998, 22, 864–872. [Google Scholar] [CrossRef]
- Watnabe, H.; Hayashi, J.; Ohzeki, H.; Moro, H.; Sugawara, M.; Eguchi, S. Biocompatibility of a silicone-coated polypropylene hollow fiber oxygenator in an in vitro model. Ann. Thorac. Surg. 1999, 67, 1315–1319. [Google Scholar] [CrossRef]
- Kaar, J.L.; Oh, H.-I.; Russell, A.J.; Federspiel, W.J. Towards improved artificial lungs through biocatalysis. Biomaterials 2007, 28, 3131–3139. [Google Scholar] [CrossRef]
- Arazawa, D.T.; Oh, H.-I.; Ye, S.-H.; Johnson, C.A., Jr.; Woolley, J.R.; Wagner, W.R.; Federspiel, W.J. Immobilized carbonic anhydrase on hollow fiber membranes accelerates CO2 removal from blood. J. Membr. Sci. 2012, 404, 25–31. [Google Scholar]
- Hunt, J.A.; Lesburg, C.A.; Christianson, D.W.; Thompson, R.B.; Fierke, C.A. Active-site Engineering of Carbonic Anhydrase and Its Applications to Biosensors. In The Carbonic Anhydrases: New horizons; Chegwidden, W.R., Carter, N.D., Edwards, Y.H., Eds.; Birkhäuser Verlag: Boston, MA, USA, 2000; pp. 221–240. [Google Scholar]
- Lindskog, S.; Nyman, P.O. Metal-binding properties of human erythrocyte carbonic anhydrase. Biochem. Biophys. Acta 1964, 85, 462–474. [Google Scholar]
- Thompson, R.B.; Jones, E.R. Enzyme-based fiber optic zinc biosensor. Anal. Chem. 1993, 65, 730–734. [Google Scholar] [CrossRef]
- Rout, G.R.; Das, P. Effect of metal toxicity on plant growth and metabolism: I. Zinc. Agronomie 2003, 23, 3–11. [Google Scholar] [CrossRef]
- Muyssen, B.T.; de Schamphelaere, K.A.; Janssen, C.R. Mechanisms of chronic waterborne zn toxicity in daphnia magna. Aquat. Toxicol. 2006, 77, 393–401. [Google Scholar] [CrossRef]
- Chen, R.F.; Kernohan, J.C. Combination of bovine carbonic anhydrase with a fluorescence sulfonamide. J. Biol. Chem. 1967, 242, 5813–5823. [Google Scholar]
- Huang, C.-C.; Lesburg, C.A.; Kiefer, L.L.; Fierke, C.A.; Christianson, D.W. Reversal of the hydrogen bond to zinc ligand histidine-119 dramatically diminishes catalysis and enhances metal equilibriation kinetics in carbonic anhydrase. Biochemistry 1996, 35, 3439–3446. [Google Scholar] [CrossRef]
- Thompson, R.B.; Ge, Z.; Patchan, M.W.; Kiefer, L.L.; Fierke, C.A. Performance enhancement of fluorescence energy transfer-based biosensors by site-directed mutagenesis of the transducer. SPIE 1996, 2508, 136–144. [Google Scholar]
- Thompson, R.B.; Patchan, M.W. Lifetime-based fluorescence energy transfer biosensing of zinc. Anal. Biochem. 1995, 227, 123–128. [Google Scholar] [CrossRef]
- Demille, G.R.; Larlee, K.; Livesey, D.L.; Mailer, K. Conformational change in carbonic anhydrase studied by perturbed directional correlations of gamma rays. Chem. Phys. Lett. 1979, 64, 534–539. [Google Scholar] [CrossRef]
- Harrington, P.C.; Wilkins, R.G. Interaction of acetazolamide and 4-nitrothiophenolate ion with bivalent metal ion derivatives of bovine carbonic anhydrase. Biochemistry 1977, 16, 448–454. [Google Scholar] [CrossRef]
- Thompson, R.B.; Ge, Z.; Patchan, M.W.; Huang, C.-C.; Fierke, C.A. Fiber optic biosensor for co(ii) and cu(ii) based on fluorescence energy transfer with an enzyme transducer. Biosens. Bioelectron. 1996, 11, 557–564. [Google Scholar] [CrossRef]
- Frederickson, C.J.; Giblin, L.J.; Krezel, A.; McAdoo, D.J.; Mueller, R.N.; Zeng, Y.; Balaji, R.V.; Masalha, R.; Thompson, R.B.; Fierke, C.A.; et al. Concentrations of extracellular free zinc (pzn)e in the central nervous system during simple anesthetization, ischemia and reperfusion. Exp. Neurol. 2006, 198, 285–293. [Google Scholar] [CrossRef]
- Thompson, R.B.; Peterson, D.; Mahoney, W.; Cramer, M.; Maliwal, B.P.; Suh, S.W.; Frederickson, C.; Fierke, C.; Herman, P. Fluorescent zinc indicators for neurobiology. J. Neurosci. Methods 2002, 118, 63–75. [Google Scholar] [CrossRef]
- Thompson, R.B.; Whetsell, W.O., Jr.; Maliwal, B.P.; Fierke, C.A.; Frederickson, C.J. Fluorescence microscopy of stimulated zn(ii) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system. J. Neurosci. Methods 2000, 96, 35–45. [Google Scholar] [CrossRef]
- Bozym, R.; Hurst, T.K.; Westerberg, N.; Stoddard, A.; Fierke, C.A.; Frederickson, C.J.; Thompson, R.B. Chapter 14 determination of zinc using carbonic anhydrase-based fluorescence biosensors. Methods Enzymol. 2008, 450, 287–309. [Google Scholar] [CrossRef]
- Wang, D.; Hurst, T.K.; Thompson, R.B.; Fierke, C.A. Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in escherichia coli. J. Biomed. Opt. 2011, 16. [Google Scholar] [CrossRef]
- McCranor, B.J.; Bozym, R.A.; Vitolo, M.I.; Fierke, C.A.; Bambrick, L.; Polster, B.M.; Fiskum, G.; Thompson, R.B. Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion. J. Bioenerg. Biomembr. 2012, 44, 253–263. [Google Scholar] [CrossRef]
- Simsek-Ege, F.A.; Bond, G.M.; Stringer, J. Matrix molecular weight cut-off for encapsulation of carbonic anhydrase in polyelectrolyte beads. J. Biomater. Sci. Polym. Ed. 2002, 13, 1175–1187. [Google Scholar] [CrossRef]
- Cowan, R.M.; Ge, J.; Qin, Y.J.; McGregor, M.L.; Trachtenberg, M.C. Co2 capture by means of an enzyme-based reactor. Ann. N.Y. Acad. Sci. 2003, 984, 453–469. [Google Scholar] [CrossRef]
- Satav, S.S.; Bhat, S.; Thayumanavan, S. Feedback regulated drug delivery vehicles: Carbon dioxide responsive cationic hydrogels for antidote release. Biomacromol 2010, 11, 1735–1740. [Google Scholar] [CrossRef]
- Han, D.; Boissiere, O.; Kumar, S.; Tong, X.; Tremblay, L.N.; Zhao, Y. Two-way co2-switchable triblock copolymer hydrogels. Macromol 2012, 45, 7440–7445. [Google Scholar] [CrossRef]
- Aggarwal, M.; Boone, C.D.; Kondeti, B.; Tu, C.; Silverman, D.N.; McKenna, R. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase ii-acetazolamide complex. Acta Crystallogr. Sect. D Biol. Crystallogr. 2013, 69, 860–865. [Google Scholar] [CrossRef]
- Sippel, K.H.; Robbins, A.H.; Domsic, J.; Genis, C.; Agbandje-McKenna, M.; McKenna, R. High-resolution structure of human carbonic anhydrase ii complexed with acetazolamide reveals insights into inhibitor drug design. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2009, 65, 992–995. [Google Scholar] [CrossRef]
- Aggarwal, M.; Kondeti, B.; McKenna, R. Insights towards sulfonamide drug specificity in alpha-carbonic anhydrases. Bioorganic Med. Chem. 2013, 21, 1526–1533. [Google Scholar] [CrossRef]
- Svastová, E.; Huliková, A.; Rafajová, M.; Zat’ovicová, M.; Gibadulinová, A.; Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C.T.; Pastorek, J.; et al. Hypoxia activates the capacity of tumor-associated carbonic anhydrase ix to acidify extracellular ph. FEBS lett. 2004, 577, 439–445. [Google Scholar] [CrossRef]
- Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.; Scozzafava, A.; Pastorekova, S.; Supuran, C.T. Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozyme ix with aromatic and heterocyclic sulfonamides. Bioorganic Med. Chem. Lett. 2003, 13, 1005–1009. [Google Scholar] [CrossRef]
- Winum, J.Y.; Rami, M.; Scozzafava, A.; Montero, J.L.; Supuran, C. Carbonic anhydrase ix: A new druggable target for the design of antitumor agents. Med. Res. Rev. 2008, 28, 445–463. [Google Scholar] [CrossRef]
- Supuran, C.T. Carbonic anhydrase inhibitors: An editorial. Expert Opin. Ther. Pat. 2013, 23, 677–679. [Google Scholar] [CrossRef]
- Supuran, C.T.; Scozzafava, A.; Casini, A. Carbonic anhydrase inhibitors. Med. Res. Rev. 2003, 23, 146–189. [Google Scholar] [CrossRef]
- Gould, S.A.; Moore, E.E.; Hoyt, D.B.; Ness, P.M.; Norris, E.J.; Carson, J.L.; Hides, G.A.; Freeman, I.H.; DeWoskin, R.; Moss, G.S. The life-sustaining capacity of human polymerized hemoglobin when red cells might be unavailable. J. Am. Coll. Surg. 2002, 195, 445–452. [Google Scholar] [CrossRef]
- Bian, Y.; Rong, Z.; Chang, T.M. Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase: A novel biotechnology-based blood substitute that transports both oxygen and carbon dioxide and also acts as an antioxidant. Artif. Cells Blood Substit. Immobil. Biotechnol. 2012, 40, 28–37. [Google Scholar] [CrossRef]
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Boone, C.D.; Habibzadegan, A.; Gill, S.; McKenna, R. Carbonic Anhydrases and Their Biotechnological Applications. Biomolecules 2013, 3, 553-562. https://doi.org/10.3390/biom3030553
Boone CD, Habibzadegan A, Gill S, McKenna R. Carbonic Anhydrases and Their Biotechnological Applications. Biomolecules. 2013; 3(3):553-562. https://doi.org/10.3390/biom3030553
Chicago/Turabian StyleBoone, Christopher D., Andrew Habibzadegan, Sonika Gill, and Robert McKenna. 2013. "Carbonic Anhydrases and Their Biotechnological Applications" Biomolecules 3, no. 3: 553-562. https://doi.org/10.3390/biom3030553