Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Qun Wan; Jerry M. Parks; B. Leif Hanson; Suzanne Zoe Fisher; Andreas Ostermann; Tobias E. Schrader; David E. Graham; Leighton Coates; Paul Langan; Andrey Kovalevsky

doi:10.1073/pnas.1504986112

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 pK_a 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 pK_a values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pK_a 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.

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.