Probing the peripheral site of human butyrylcholinesterase

Abstract

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) catalyze the hydrolysis of the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. For both enzymes, hydrolysis takes place near the bottom of a 20 Å deep active site gorge. A number of amino acid residues within the gorge have been identified as important in facilitating efficient catalysis and inhibitor binding. Of particular interest is the catalytic triad, consisting of serine, histidine, and glutamate residues, that mediates hydrolysis. Another site influencing the catalytic process is located above the catalytic triad toward the periphery of the active site gorge. This peripheral site (P-site) contains a number of aromatic amino acid residues as well as an aspartate residue that is able to interact with cationic substrates and guide them down the gorge to the catalytic triad. In human AChE, certain aryl residues in the vicinity of the anionic aspartate residue (D74), such as W286, have been implicated in ligand binding and have therefore been considered part of the P-site of the enzyme. The present study was undertaken to explore the P-site of human BuChE and determine whether, like AChE, aromatic side chains near the peripheral aspartate (D70) of this enzyme contribute to ligand binding. Results obtained, utilizing inhibitor competition studies and BuChE mutant species, indicate the participation of aryl residues (F329 and Y332) in the E-helix component of the BuChE active site gorge, along with the anionic aspartate residue (D70), in binding ligands to the P-site of the enzyme.

Figure 1

Active site gorge with homologous residues shown for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The crystal structures of human AChE (PDB ID: 1B41) and BuChE (PDB ID: 1POI) were obtained from the Protein Data Bank, and PyMol was employed to delete all amino acids save for those selected residues found in the active site.

Figure 2

Structures of cholinesterase inhibitors used.

Figure 3

Plots of average second-order substrate hydrolysis ratios by BuChE in the absence (k_E[I]=0) and presence (k_E+I) of thioflavin T (1) or propidium (2). These plots show a linear relationship between the reciprocal of the second-order hydrolysis rate and inhibitor concentration.

Figure 4

Plots of second-order substrate hydrolysis rates by BuChE or AChE with thioflavin T and in the presence (triangle) or absence (circle) of another inhibitor with lines fitted or calculated according to eq 4. Dotted lines represent the theoretical plot that denotes complete competition between the inhibitor and thioflavin T for that enzyme. For example, for BuChE, propidium and thioflavin T do not compete as exemplified by overlap in the plots with and without propidium. In contrast, for AChE, propidium competes with thioflavin T as indicated by absence of overlap in the plots with and without propidium, and overlap between the presence of propidium and complete competition plots.

Figure 5

Enzyme activity of wild type BuChE and its mutants in the absence and presence of compounds 1–5. The % residual enzyme activity indicates the activity in the presence of inhibitor relative to the activity in the absence of inhibitor for each mutant. Note that D70 mediates, in part, inhibition by all cationic inhibitors (compounds 1–4). In addition, residues of the E-helix, F329 and Y332, are involved in ligand binding (compounds 2–5). Thus, D70, Y332, and F329 are components of the P-site of BuChE.