Butyrylcholinesterase active site

To help the reader gain a better understanding of the three-dimensional structure of the catalytic site of an esterase, Fig. 3.8 presents the 3D structure of human butyrylcholinesterase (EC 3.1.1.8) obtained by homology modeling [42], The overall structure of the enzyme is shown in Fig. 3.8, a, while Fig. 3.8,b shows a closeup of the active site with the catalytic triad highlighted and the close spatial relationship of the Ser-His-Glu residues revealed. 

Acetylcholinesterase is the primary target of these drugs, but butyrylcholinesterase is also inhibited. Acetylcholinesterase is an extremely active enzyme. In the initial catalytic step, acetylcholine binds to the enzyme s active site and is hydrolyzed, yielding free choline and the acetylated enzyme. In the second step, the covalent acetyl-enzyme bond is split, with the addition of water (hydration). The entire process occurs in approximately 150 microseconds. 

Fidder and coworkers (50) developed a versatile procedure that identifies phosphylated butyrylcholi-nesterase. Adducted butyrylcholinesterase is isolated from plasma by affinity chromatography (procainamide column), digested with pepsin, and a nonapep-tide containing the phosphylated active-site serine residue detected using LC/ESI/MS/MS (quadrupole-TOF hybrid instrument). A C18 150 x 0.3-mm LC column was used, eluted with a gradient of water-acetonitrile-0.2 % formic acid. The method was applied successfully to casualties of sarin poisoning from the Tokyo subway attack (see Chapter 17). 

Saxena, A., Redman, A.M.G., Jiang, X., Lockridge, O., Doctor, B.P. (1999). Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase. Chem. Biol. Interact. 119-20 61-9. 

Fidder et al. introduced an electrospray-ionization tandem mass spectrometry method for diagnosing OP exposure by measuring the mass of the OP-labeled active site peptide of human butyrylcholinesterase (Fidder et al, 2002). His starting material was 0.5 ml of human plasma from a victim of the Tokyo subway attack. The mass of the active site peptide was higher by 120 atomic mass units, compared to the mass of the unlabeled active site peptide. This added mass was exactly the added mass expected from sarin. The peptide s MS-MS fragmentation spectrum yielded the sequence of the peptide, and verified that the OP label was on serine 198, the active site serine. Examples of the MS-MS spectra from tryptic peptides of pure, OP-labeled human butyrylcholinesterase are shown in Figure 56.1. 

FIGURE 57.9. Inhibition and aging of serine esterases by diisopropylphosphorofluoridate (DFP). The active site serine is organophosphorylated in the inhibition step. Aging results in net loss of an isopropyl group to yield the monoisopropylphosphoryl esterase. This mode of inhibition and aging has been established for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and neuropathy target esterase catalytic domain (NEST) (Kropp and Richardson, 2007). 

Doom, J.A., Talley, T.T., Thompson, C.M., Richardson, R.J. (2001a). Probing the active sites of butyrylcholinesterase and cholesterol esterase with isomalathion conserved stereoselective inactivation of serine hydrolases stmcturally related to acetylcholinesterase. Chem. Res. Toxicol. 14 807-13. 

A series of novel l, 3-dihydroxyxanthone Mannich base derivatives were synthesized, structure elucidated, and evaluated for anticholinesterase activity. Most of the target compounds exhibited moderate to good inhibitory activities with the IC50 values at micromole level concentration against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Among them, 2 - ((diethylamino )methyl) -1 -hydroxy- 3 - (3 -methylbut-2-enyloxy) -9H-xanthen-9-one showed potent inhibitory activity against AChE and the best inhibitory activity against BuChE. The Mannich base derivatives were likely to bind to the active site (AS) and the peripheral anionic site (PAS) of cholinesterases [169]. 

The generality of this new type of shape-activity correlation is demonstrated for five receptor/substrate systems trypsin/arylammonium inhibitors the D2-dop-amine receptor/dopamine derivative agonists trypsin/organophosphate inhibitors acetylcholinesterase/organophosphates and butyrylcholinesterase/organo-phosphates. The correlations were obtained both for active-site induced chiral conformers and for inherently chiral inhibitors. Interestingly, for some of these cases the correlation of activity with structure is hidden when classical parameters, such as chain length, are taken, but is revealed with this shape descriptor. 

FIGURE 8.4 A ball-and-stick computer model of the active site of the double mutant of butyrylcholinesterase G117H/E197Q. In addition to the His 117 and Gin 197, the active site triad amino acid residues of His 438, Ser 198, and Glu 325 are also depicted with soman at the active site. The distances between the phosphorus atom of soman and His 117 is 5.05 A and distance between the phosphorus atom of soman and the active site His 438 is 5.94 A. (Data are from Millard et al., 1998.)... 

The basis of our current understanding of the two types of active site which are present in cholinesterase has been provided by Wilson and Bergmann (W30), who substantiated the concept of anionic and esteratic sites introduced by Zeller and Bisegger (Z2). Although it is now generally accepted that the hydrolysis of the ester bond of the substrate occurs at the esteratic site, there is still some uncertainty regarding the existence of the anionic site in butyrylcholinesterase an alternative site has been proposed by Augustinsson (A28). 

Comprehensive reviews (Kl, Ul) of the active sites of cholinesterase both postulated the presence not only of an esteratic site for butyrylcholinesterase but also of an anionic site. Additionally, in the region of the anionic site, there are two hydrophobic areas, one directly surrounding the anionic group and the second located at some distance from it (Kl). The presence of hydrophobic areas has been established (B32, C3, H29, H45, MIO) by the use of fluorescent probes with spectral responses which reflect the environment of the probe. Such probes can be used to monitor changes in the conformations of enzymes and can be designed to be active-site-directed, competitive inhibitors (H30). Aspects of the spectroscopy of intrinsic and extrinsic fluorescent probes have been reported (C3). 

Irreversible inhibitors are effectively esteratic site inhibitors which, like true substrates, react with the hydroxyl group of serine at the catalytic active site. Such inhibitors, sometimes referred to as acid-transferring inhibitors, include the organophosphates, the organo-sulfonates, and the carbamates. All form acyl-enzyme complexes which, unlike substrate-enzyme intermediates, are relatively stable to hydrolysis. Indeed, the phosphorylated enzyme intermediates have half-lives from a few hours to several days (A12), whereas the sulfonated or carbamylated enzyme complexes have much shorter half-lives—several minutes to a few hours. Several strong lines of direct evidence point to the formation of an acyl complex—the isolation of phosphorylated serine from hydrolysates of horse cholinesterase (J2), complex formation and carbamylation (02), and the sulfonation of butyrylcholinesterase by methanesulfonyl fluoride in the presence of tubocurarine and eserine (P6). 

Several piezoelectric sensors for the detection of low molecular weight compounds like pesticides (9-11), toxins (12), or drugs (13) have been reported in recent years (Table 1). A piezoelectric biosensor with a reversibly coupled (via His-tag) paraoxon for the binding of tetrameric butyrylcholinesterase to the active site of the enzyme has also been described by us (14). The commercially available devices have recently been reviewed (15, 16). 

The hydrophobic area surrounding the anionic site plays a more important role for butyrylcholinesterase than for acetylcholinesterase. The greater importance of this hydrophobic area for butyrylcholinesterase could help to explain and resolve some of the opposing views of earlier workers (A26). Kabachnik et al. (Kl) also proposed that in the vicinity of the esteratic site of butyrylcholinesterase there are two hydrophobic areas separated by a hydrophilic group. Differences in length and structure of the hydrophobic areas of the active surfaces of butyryl- and... 

---