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. 1972 Sep;69(9):2422–2425. doi: 10.1073/pnas.69.9.2422

H2CO3 as Substrate for Carbonic Anhydrase in the Dehydration of HCO3-

Seymour H Koenig 1, Rodney D Brown III 1
PMCID: PMC426955  PMID: 4627028

Abstract

Carbonic anhydrase, a metalloenzyme containing one zinc atom per protein molecule of molecular weight 30,000, catalyzes the interconversion of CO2 and HCO3- in solution. The rate of catalysis, among the fastest known, is pH-dependent, with a pKEnz near neutral. Arguments are presented to show that: (i) only the high-pH form of the enzyme is active both for the hydration and dehydration reactions (ii) at high pH there is an H2O ligand on the metal (not an OH- as is often argued), and (iii) the substrate for the dehydration reaction is the neutral H2CO3 molecule. The arguments are based on data in the literature on the nuclear relaxation rates of Cl- ions and water protons in solutions of carbonic anhydrase, on strict application of the principle of microscopic reversibility, and on kinetic considerations. It has been argued that H2CO3 cannot be the substrate for the dehydration reaction because the observed CO2 production rate is somewhat faster than the maximum rate at which H2CO3 molecules can diffuse to the active site of the enzyme. However, current models that consider HCO3- as the substrate implicity require that protons diffuse to the enzyme at an even greater rate, well outside the limitations imposed by diffusion. We consider two mechanisms to obviate the diffusion limitation problem, and conjecture that at high substrate concentration, H2CO3 reaches the active site by collision with the enzyme molecule, and subsequent surface diffusion to the active site. At lower substrate concentrations, corresponding to [HCO3-] <1 mM, generation of H2CO3 molecules near the enzyme by the recombination reaction H+ + HCO3- → H2CO3 can supply an adequate flux of substrate to the active site.

Keywords: metalloenzymes, enzyme mechanism, CO2 hydration

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Coleman J. E. Mechanism of action of carbonic anhydrase. Subtrate, sulfonamide, and anion binding. J Biol Chem. 1967 Nov 25;242(22):5212–5219. [PubMed] [Google Scholar]
  2. Fabry M. E., Koenig S. H., Schillinger W. E. Nuclear magnetic relaxation dispersion in protein solutions. IV. Proton relaxation at the active site of carbonic anhydrase. J Biol Chem. 1970 Sep 10;245(17):4256–4262. [PubMed] [Google Scholar]
  3. Garg L. C., Maren T. H. The rates of hydration of carbon dioxide and dehydration of carbonic acid at 37 degrees. Biochim Biophys Acta. 1972 Jan 28;261(1):70–76. doi: 10.1016/0304-4165(72)90315-7. [DOI] [PubMed] [Google Scholar]
  4. KERNOHAN J. C. THE PH-ACTIVITY CURVE OF BOVINE CARBONIC ANHYDRASE AND ITS RELATIONSHIP TO THE INHIBITION OF THE ENZYME BY ANIONS. Biochim Biophys Acta. 1965 Feb 22;96:304–317. [PubMed] [Google Scholar]
  5. Khalifah R. G., Edsall J. T. Carbon dioxide hydration activity of carbonic anhydrase: kinetics of alkylated anhydrases B and C from humans (metalloenzymes-isoenzymes-active sites-mechanism). Proc Natl Acad Sci U S A. 1972 Jan;69(1):172–176. doi: 10.1073/pnas.69.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Khalifah R. G. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J Biol Chem. 1971 Apr 25;246(8):2561–2573. [PubMed] [Google Scholar]
  7. Koenig S. H., Schillinger W. E. Nuclear magnetic relaxation dispersion in protein solutions. I. Apotransferrin. J Biol Chem. 1969 Jun 25;244(12):3283–3289. [PubMed] [Google Scholar]
  8. LINDSKOG S. Effects of pH and inhibitors on some properties related to metal binding in bovine carbonic anhydrase. J Biol Chem. 1963 Mar;238:945–951. [PubMed] [Google Scholar]
  9. Lindskog S. Interaction of cobalt(II)--carbonic anhydrase with anions. Biochemistry. 1966 Aug;5(8):2641–2646. doi: 10.1021/bi00872a023. [DOI] [PubMed] [Google Scholar]
  10. Magid E. The dehydration kinetics of human erythrocytic carbonic anhydrases B and C. Biochim Biophys Acta. 1968 Jan 8;151(1):236–244. doi: 10.1016/0005-2744(68)90178-2. [DOI] [PubMed] [Google Scholar]
  11. Taylor P. W., Burgen A. S. Kinetics of carbonic anhydrase-inhibitor complex formation. a comparison of anion- and sulfonamide-binding mechanisms. Biochemistry. 1971 Oct 12;10(21):3859–3866. doi: 10.1021/bi00797a010. [DOI] [PubMed] [Google Scholar]
  12. Taylor P. W., Feeney J., Burgen A. S. Investigation of the mechanism of ligand binding with cobalt(II) human carbonic anhydrase by 1 H and 19 F nuclear magnetic resonance spectroscopy. Biochemistry. 1971 Oct 12;10(21):3866–3875. doi: 10.1021/bi00797a011. [DOI] [PubMed] [Google Scholar]
  13. Taylor P. W., King R. W., Burgen A. S. Influence of pH on the kinetics of complex formation between aromatic sulfonamides and human carbonic anhydrase. Biochemistry. 1970 Sep 29;9(20):3894–3902. doi: 10.1021/bi00822a007. [DOI] [PubMed] [Google Scholar]
  14. Ward R. L. 35C1 nuclear magnetic resonance studies of a zinc metalloenzyme carbonic anhydrase. Biochemistry. 1969 May;8(5):1879–1883. doi: 10.1021/bi00833a016. [DOI] [PubMed] [Google Scholar]
  15. Ward R. L., Fritz K. J. 35Cl-NMR studies of Co2+ carbonic anhydrases. Biochem Biophys Res Commun. 1970 May 22;39(4):707–711. doi: 10.1016/0006-291x(70)90262-7. [DOI] [PubMed] [Google Scholar]
  16. Ward R. L. Zinc environmental differences in carbonic anhydrase isozymes. Biochemistry. 1970 Jun 9;9(12):2447–2454. doi: 10.1021/bi00814a009. [DOI] [PubMed] [Google Scholar]

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