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 [(13)C]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.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545;
authors.forename: Ryszard
authors.initials: R
authors.lastname: Michalczyk
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545;
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Unkefer
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545;
authors.forename: John-Paul
authors.initials: JP
authors.lastname: Bacik
authors.affiliation: Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, 85747 Garching, Germany;
authors.forename: Tobias E
authors.initials: TE
authors.lastname: Schrader
authors.affiliation: Heinz Maier-Leibnitz Zentrum (MLZ), Technische Universität München, 85748 Garching, Germany;
authors.forename: Andreas
authors.initials: A
authors.lastname: Ostermann
authors.affiliation: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge TN 37831; and.
authors.forename: Andrey Y
authors.initials: AY
authors.lastname: Kovalevsky
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610.
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545; zoe.fisher@esss.se.
authors.forename: Suzanne Zoë
authors.initials: SZ
authors.lastname: Fisher
mesh_terms: D002256:Carbonic Anhydrases / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D018360:Crystallography, X-Ray / Q000379:methods*; D004798:Enzymes / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009502:Neutrons; D011487:Protein Conformation; D011522:Protons; D055672:Static Electricity; D014443:Tyrosine / Q000737:chemistry*; D014867:Water / Q000737:chemistry
chemical_list: D002412:Cations; D004798:Enzymes; D011522:Protons; D014867:Water; D014443:Tyrosine; D002256:Carbonic Anhydrases
keywords: neutron crystallography; perturbed pKa; proton transfer; solution NMR