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

Ryszard Michalczyk, Clifford J. Unkefer, John Paul Bacik, Tobias E. Schrader, Andreas Ostermann, Andrey Y. Kovalevsky, Robert McKenna, Suzanne Zoë Fisher

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

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 hydrogenbonding interactions.

Original languageEnglish
Pages (from-to)5673-5678
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number18
DOIs
StatePublished - May 5 2015

Keywords

  • Neutron crystallography
  • Perturbed pKa
  • Proton transfer
  • Solution NMR

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