Dynamic interchange between two protonation states is characteristic of active sites of cholinesterases

Abstract

Cholinesterases are well-known and widely studied enzymes crucial to human health and involved in neurology, Alzheimer's, and lipid metabolism. The protonation pattern of active sites of cholinesterases influences all the chemical processes within, including reaction, covalent inhibition by nerve agents, and reactivation. Despite its significance, our comprehension of the fine structure of cholinesterases remains limited. In this study, we employed enhanced-sampling quantum-mechanical/molecular-mechanical calculations to show that cholinesterases predominantly operate as dynamic mixtures of two protonation states. The proton transfer between two non-catalytic glutamate residues follows the Grotthuss mechanism facilitated by a mediator water molecule. We show that this uncovered complexity of active sites presents a challenge for classical molecular dynamics simulations and calls for special treatment. The calculated proton transfer barrier of 1.65 kcal/mol initiates a discussion on the potential existence of two coupled low-barrier hydrogen bonds in the inhibited form of butyrylcholinesterase. These findings expand our understanding of structural features expressed by highly evolved enzymes and guide future advances in cholinesterase-related protein and drug design studies.

Keywords: LBHB; QM/MM; biomolecular simulations; cholinesterase; enzymology; free energy profile; proton rearrangements.

FIGURE 1

Active site structure and reaction mechanisms of cholinesterases. (a) Active site scheme of human cholinesterases showing catalytic and glutamate triads as shared features. One of a number of hypothesized protonation patterns is shown for demonstration purposes. (b and c) Active site structure of (b) hBChE and (c) hAChE showing spatial arrangements of discussed features. (d) Schemes of principle reactions catalyzed by cholinesterases. Acylation and deacylation are consecutive stages of the reaction with native substrates. Self‐reactivation is only observed for modified variants including G117H hBChE. Reactivation by oximes is not shown. hAChE, human acetylcholinesterase; hBChE, human butyrylcholinesterase.

FIGURE 2

Reference and distorted geometries of hBChE extended active site. (a) Reference geometry and h‐bonding network for the free form (PDB ID 6QAA). (b) Reference geometry and h‐bonding network for the OP‐inhibited form (PDB ID 1XLW). (c) dRMSD values for h‐bonding networks after 100 ns MD simulations. For time series, refer to Figures S1 and S2 in Data S1. Gray lines represent comparison statistics obtained under position restraints. (d–i) Snapshots of best replicas from MD simulations. Labeled are residues that moved the most. White dashes represent selected lost contacts. Yellow dashes represent new interactions that should not have been formed. (d–f) Free form. (g–i) DEP‐inhibited form. (d and g) Glu197/Glu441 systems. (e and h) Glh197/Glu441 systems. (f and i) Glu197/Glh441 systems. Nonpolar hydrogens are omitted for clarity. DEP, diethyl‐phosphorylated; dRMSD, distance RMSD; hBChE, human butyrylcholinesterase; MD, molecular dynamics; OP, organophosphate.

FIGURE 3

Division of the hBChE active site into proton depots. (a) H‐bonds along which a proton transfer may happen. (b) E441 interacts with the water molecule exclusively by its upper oxygen atom as seen from the electron density map. (c) Formalization of terms used to set up computational experiments. a1–6 and b1–6 represent distances to hydrogens that may be present within the depots. These distances are used to construct collective variables for metadynamics simulations (see Tables S1 and S2 in Data S1). hBChE, human butyrylcholinesterase.

FIGURE 4

Systematic scanning of all protonation possibilities for DEP‐hBChE with QM/MM metadynamics yields free energy profiles that guide the selection of the most promising arrangements. Numbers in circles represent the assigned indices of selected candidate states. (a–c) No protons in depot 1. (d–f) 1 proton in depot 1. (g and h). Two protons in depot 1. (a, d, and g) No protons in depot 2. (b, e, and h) One proton in depot 2. (c and f) Two protons in depot 2. Color maps represent free energies in kcal/mol. One color level is 1 kcal/mol, one outline is 2 kcal/mol. Collective variables are detailed in Figure 3 and in Supporting information (Tables S1 and S2 in Data S1). Representative structures for each system are shown in Figure 5. On 1‐D plots, standard errors of the mean are shown as shaded areas. DEP‐hBChE, diethyl‐phosphorylated human butyrylcholinesterase; QM/MM, quantum‐mechanical/molecular‐mechanical.

FIGURE 5

Shortlisted protonation patterns for DEP‐hBChE. Numbers in circles represent the assigned state indices used in Figure 4 and throughout the work. (a) 0 additional protons in both depot 1 and 2. (b) 0 protons in depot 1 and 1 proton in depot 2. (c) 0 protons in depot 1 and 2 protons in depot 2. (d) 1 proton in depot 1 and 0 protons in depot 2. (e) 1 proton in each depot. (f) 1 proton in depot 1 and 2 protons in depot 2. (g) 2 protons in depot 1 and 0 protons in depot 2. (h) 2 protons in depot 1 and 1 proton in depot 2. (i) 2 protons in each depot. Nonpolar hydrogens are omitted for clarity. DEP‐hBChE, diethyl‐phosphorylated human butyrylcholinesterase.

FIGURE 6

Identification of the protonation state of DEP‐hBChE. (a) Retention of the reference active site structure in QM/MM MD for all selected systems. (b) Schemes of systems 9 and 10 that populate the correct DEP‐hBChE ensemble. (c) 2‐d free energy profile of the proton transfer, DFTB3/MM. (d) Comparison of DFTB3‐D4/MM and PBE/DZVP‐MOLOPT free energy profiles. Shaded areas represent standard errors of the mean. DEP‐hBChE, diethyl‐phosphorylated human butyrylcholinesterase; MD, molecular dynamics; QM/MM, quantum‐mechanical/molecular‐mechanical.

FIGURE 7

Preferences for internal or external Glh differ between hAChE and hBChE, in reaction and inhibition stages and with the substitution of the key active site residue. (a) Free energy differences and barriers for the proton transfer in stable states along the reaction and inhibition processes. The X axis below 0 indicates the preference for Glh197. (b) Differences in the proton hopping behavior of WT and mutated hAChE, hBChE, and tcAChE in the free form. Reciprocal N ↔ V substitutions soften the differences between the enzymes. Error bars represent standard errors of the mean; some bars are smaller than markers and are overlaid. Exact values can be found in Table S3 in Data S1, profiles in Figures S8–11 in Data S1. (c–e) Differences in the internal Glu interactions in three cholinesterases. (c) hBChE. (d) hAChE. (e) tcAChE. White dashes represent the absence of the interaction. hAChE, human acetylcholinesterase; hBChE, human butyrylcholinesterase.

FIGURE 8

Differences in the water‐binding geometry of the DEP‐hBChE active site under different treatments. For ff99SB‐ildn, the Glh441 system was used. Gray dashes represent measurements from the 1XLW x‐ray structure. DEP‐hBChE, diethyl‐phosphorylated human butyrylcholinesterase.