A collection of research articles explores developments in interfacial transport and mixing, with wide-ranging practical applications.
New Research In
Physical Sciences
Featured Portals
Articles by Topic
Biological Sciences
Featured Portals
Articles by Topic
Edited by Dagmar Ringe, Brandeis University, Waltham, MA, and accepted by the Editorial Board August 18, 2015 (received for review March 12, 2015)
Significance
Most enzymatic reactions involve hydrogen or proton transfer among the enzyme, substrate, and water at physiological pH. Thus, enzyme catalysis cannot be fully understood without accurate mapping of hydrogen atom positions in these macromolecular catalysts. Direct information on the location of hydrogen atoms can be obtained using neutron crystallography. We used neutron crystallography and biomolecular simulation to characterize the initial stage of the glycoside hydrolysis reaction catalyzed by a family 11 glycoside hydrolase. We provide evidence that the catalytic glutamate residue alternates between two conformations bearing different basicities, first to obtain a proton from the bulk solvent, and then to deliver it to the glycosidic oxygen to initiate the hydrolysis reaction.
Abstract
Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.
- glycoside hydrolase
- protonation state
- macromolecular neutron crystallography
- xylanase
- molecular simulations
Footnotes
- ↵1To whom correspondence should be addressed. Email: kovalevskyay{at}ornl.gov.
Author contributions: Q.W., D.E.G., P.L., and A.K. designed research; Q.W., J.M.P., and A.K. performed research; Q.W., J.M.P., B.L.H., S.Z.F., A.O., T.E.S., L.C., and A.K. analyzed data; Q.W., J.M.P., D.E.G., P.L., and A.K. wrote the paper; B.L.H. collected X-ray diffraction data; and S.Z.F., A.O., T.E.S., and L.C. collected neutron diffraction data.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. D.R. is a guest editor invited by the Editorial Board.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4S2H, 4S2G, 4S2F, 4S2D, 4XPV, 4XQD, 4XQ4, and 4XQW).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1504986112/-/DCSupplemental.
- Qun Wana,
- Jerry M. Parksb,
- B. Leif Hansonc,
- Suzanne Zoe Fisherd,
- Andreas Ostermanne,
- Tobias E. Schraderf,
- David E. Grahamg,
- Leighton Coatesh,
- Paul Langanh, and
- Andrey Kovalevskyh,1
- aDepartment of Physics, College of Science, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China;
- bUniversity of Tennessee/Oak Ridge National Laboratory Center for Molecular Biophysics, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831;
- cChemistry Department, University of Toledo, Toledo, OH 43606;
- dScientific Activities Division, European Spallation Source, Lund 22100, Sweden;
- eHeinz Maier-Leibnitz Zentrum, Technische Universität München, 85748 Garching, Germany;
- fJülich Centre for Neutron Science at Heinz Maier-Leibnitz Zentrum, Forschungszentrum Jülich GmbH, 85747 Garching, Germany;
- gBiosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831;
- hBiology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831
Edited by Dagmar Ringe, Brandeis University, Waltham, MA, and accepted by the Editorial Board August 18, 2015 (received for review March 12, 2015)
Significance
Most enzymatic reactions involve hydrogen or proton transfer among the enzyme, substrate, and water at physiological pH. Thus, enzyme catalysis cannot be fully understood without accurate mapping of hydrogen atom positions in these macromolecular catalysts. Direct information on the location of hydrogen atoms can be obtained using neutron crystallography. We used neutron crystallography and biomolecular simulation to characterize the initial stage of the glycoside hydrolysis reaction catalyzed by a family 11 glycoside hydrolase. We provide evidence that the catalytic glutamate residue alternates between two conformations bearing different basicities, first to obtain a proton from the bulk solvent, and then to deliver it to the glycosidic oxygen to initiate the hydrolysis reaction.
Abstract
Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.
- glycoside hydrolase
- protonation state
- macromolecular neutron crystallography
- xylanase
- molecular simulations
Footnotes
- ↵1To whom correspondence should be addressed. Email: kovalevskyay{at}ornl.gov.
Author contributions: Q.W., D.E.G., P.L., and A.K. designed research; Q.W., J.M.P., and A.K. performed research; Q.W., J.M.P., B.L.H., S.Z.F., A.O., T.E.S., L.C., and A.K. analyzed data; Q.W., J.M.P., D.E.G., P.L., and A.K. wrote the paper; B.L.H. collected X-ray diffraction data; and S.Z.F., A.O., T.E.S., and L.C. collected neutron diffraction data.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. D.R. is a guest editor invited by the Editorial Board.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4S2H, 4S2G, 4S2F, 4S2D, 4XPV, 4XQD, 4XQ4, and 4XQW).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1504986112/-/DCSupplemental.
Protonation states in a glycoside hydrolase
Sign up for Article Alerts
Article Classifications
- Biological Sciences
- Biophysics and Computational Biology
Jump to section
You May Also be Interested in
Quantum computers capture headlines, but a quieter revolution in the world of sensors may be just as profound.
Image credit: Ola J. Joensen (Niels Bohr Institute, Copenhagen, Denmark).
Opinion: To advance sustainable stewardship, we must document not only biodiversity but geodiversity
Key abiotic features and processes should be incorporated into policy documents.
Image credit: Shutterstock/1968.
Researchers report a marine ammonite from the Cretaceous Period preserved in Burmese amber from Myanmar, and the rare specimen in amber warrants exploration of the possibly exceptional circumstances of its fossilization.
Image courtesy of Bo Wang.
Exposure to low humidity makes mice infected with influenza more susceptible to severe disease by impairing airway tissue repair and innate antiviral defense, suggesting that controlling humidity may help curb influenza during winter.
Image courtesy of Pixabay/qimono.