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 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.
        
Title: Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates Masson P, Xie W, Froment MT, Levitsky V, Fortier PL, Albaret C, Lockridge O Ref: Biochimica & Biophysica Acta, 1433:281, 1999 : PubMed
Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.