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Title: Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer

Abstract

Human carbonic anhydrase II (HCA II) uses a Zn-bound OH-/H2O mechanism to catalyze the reversible hydration of CO2. This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pKa compared with free tyrosine. To further probe the pKa) of this residue, NMR spectroscopic measurements of [13C] Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pKa of Tyr7 and how pH and Tyr proximity to Zn affect hydrogen-bonding interactions.

Authors:
 [1];  [1];  [1];  [2];  [3];  [4];  [5];  [6]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  2. Julich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Garching (Germany)
  3. Technische Univ. Munchen, Garching (Germany)
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  5. Univ. of Florida, Gainesville, FL (United States)
  6. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Julich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Garching (Germany)
Publication Date:
Research Org.:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1236701
Alternate Identifier(s):
OSTI ID: 1261433
Report Number(s):
LA-UR-15-20968
Journal ID: ISSN 0027-8424
Grant/Contract Number:  
AC52-06NA25396; AC05-00OR22725; 20110535ER
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 18; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; crystallography; NMR spectroscopy; proton transfer; neutron crystallography; perturbed pKa; solution NMR; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 36 MATERIALS SCIENCE

Citation Formats

Michalczyk, Ryszard, Unkefer, Clifford J., Bacik, John -Paul, Schrader, Tobias E., Ostermann, Andreas, Kovalevsky, Andrey Y., McKenna, Robert, and Fisher, Suzanne Zoe. Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer. United States: N. p., 2015. Web. doi:10.1073/pnas.1502255112.
Michalczyk, Ryszard, Unkefer, Clifford J., Bacik, John -Paul, Schrader, Tobias E., Ostermann, Andreas, Kovalevsky, Andrey Y., McKenna, Robert, & Fisher, Suzanne Zoe. Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer. United States. https://doi.org/10.1073/pnas.1502255112
Michalczyk, Ryszard, Unkefer, Clifford J., Bacik, John -Paul, Schrader, Tobias E., Ostermann, Andreas, Kovalevsky, Andrey Y., McKenna, Robert, and Fisher, Suzanne Zoe. 2015. "Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer". United States. https://doi.org/10.1073/pnas.1502255112. https://www.osti.gov/servlets/purl/1236701.
@article{osti_1236701,
title = {Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer},
author = {Michalczyk, Ryszard and Unkefer, Clifford J. and Bacik, John -Paul and Schrader, Tobias E. and Ostermann, Andreas and Kovalevsky, Andrey Y. and McKenna, Robert and Fisher, Suzanne Zoe},
abstractNote = {Human carbonic anhydrase II (HCA II) uses a Zn-bound OH-/H2O mechanism to catalyze the reversible hydration of CO2. This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pKa compared with free tyrosine. To further probe the pKa) of this residue, NMR spectroscopic measurements of [13C] Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pKa of Tyr7 and how pH and Tyr proximity to Zn affect hydrogen-bonding interactions.},
doi = {10.1073/pnas.1502255112},
url = {https://www.osti.gov/biblio/1236701}, journal = {Proceedings of the National Academy of Sciences of the United States of America},
issn = {0027-8424},
number = 18,
volume = 112,
place = {United States},
year = {Tue May 05 00:00:00 EDT 2015},
month = {Tue May 05 00:00:00 EDT 2015}
}

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