CO2 + H2O -->HCO3 + H+. This catalysis involves attack on CO2 by zinc-bound hydroxide followed by rate-limiting proton transfers from the active site to solution to regenerate the zinc-bound hydroxide. Three genetically distinct forms of CA in the 1-, ss-, and 3-classes utilize an amino-acid as an intramolecular proton shuttle;this residue accepts protons from the zinc-bound water through a network of hydrogen-bonded waters at a turnover rate as great as 106 s-1 and transfers them to solution. The unifying goal of this application is to expand our use of the carbonic anhydrases to understand such rate-limiting and long-range proton transfer steps in a way that can be extended to other proteins. A concurrent goal is to understand the proton transfers in carbonic anhydrase and to elucidate the significance of free energy plots for proton transfer in an enzyme or protein. We will use site-specific mutagenesis to place proton transfer groups at strategic locations in enzymes from the three classes of CA. This provides a range of catalytic activities, geometries, and active-site environments. Stopped-flow spectrophotometry, 18O exchange between CO2 and water measured by mass spectrometry, and solvent H/D isotope effects will be used to investigate rate constants for intramolecular proton transfer. Crystal structures of important mutants will be determined by X-ray and neutron diffraction, and we will attempt to observe hydrogen atoms in proton transfer pathways. We will determine specifically how distances, location, and environment in a protein influence the rate of proton transfer through intervening solvent structures. We will apply Marcus rate theory to determine and interpret the intrinsic energy barriers and thermodynamic components for the proton transfers and relate them to the structural and chemical features of the CA active site.

Public Health Relevance

This research probes the frontiers of the very basic steps of proton transfer in biological systems. These steps are an essential component of energy generating mechanisms in all cells. These investigations will also show how carbonic anhydrase functions in catalysis. New forms of this enzyme are associated with malignant tumors and appear to promote growth and metastasis. Knowing how carbonic anhydrase works will provide better medications to inhibit this enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025154-32
Application #
7777790
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
1978-04-01
Project End
2013-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
32
Fiscal Year
2010
Total Cost
$344,337
Indirect Cost
Name
University of Florida
Department
Pharmacology
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Kim, Chae Un; Song, HyoJin; Avvaru, Balendu Sankara et al. (2016) Tracking solvent and protein movement during CO2 release in carbonic anhydrase II crystals. Proc Natl Acad Sci U S A 113:5257-62
Zhu, Wen; Easthon, Lindsey M; Reinhardt, Laurie A et al. (2016) Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase. Biochemistry 55:2163-73
Mahon, Brian P; Bhatt, Avni; Socorro, Lilien et al. (2016) The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis. Biochemistry 55:4642-53
Díaz-Torres, Natalia A; Mahon, Brian P; Boone, Christopher D et al. (2015) Structural and biophysical characterization of the ?-carbonic anhydrase from the gammaproteobacterium Thiomicrospira crunogena XCL-2: insights into engineering thermostable enzymes for CO2 sequestration. Acta Crystallogr D Biol Crystallogr 71:1745-56
Pinard, Melissa A; Mahon, Brian; McKenna, Robert (2015) Probing the surface of human carbonic anhydrase for clues towards the design of isoform specific inhibitors. Biomed Res Int 2015:453543
Aggarwal, Mayank; Chua, Teck Khiang; Pinard, Melissa A et al. (2015) Carbon Dioxide ""Trapped"" in a ?-Carbonic Anhydrase. Biochemistry 54:6631-8
Mahon, Brian P; Pinard, Melissa A; McKenna, Robert (2015) Targeting carbonic anhydrase IX activity and expression. Molecules 20:2323-48
Boone, Christopher D; Rasi, Valerio; Tu, Chingkuang et al. (2015) Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II. FEBS J 282:1445-57
Mahon, Brian P; Lomelino, Carrie L; Salguero, Antonieta L et al. (2015) Observed surface lysine acetylation of human carbonic anhydrase II expressed in Escherichia coli. Protein Sci 24:1800-7
Arazawa, D T; Kimmel, J D; Finn, M C et al. (2015) Acidic sweep gas with carbonic anhydrase coated hollow fiber membranes synergistically accelerates CO2 removal from blood. Acta Biomater 25:143-9

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