Butyrylcholinesterase substrates

Thioesters play a paramount biochemical role in the metabolism of fatty acids and lipids. Indeed, fatty acyl-coenzyme A thioesters are pivotal in fatty acid anabolism and catabolism, in protein acylation, and in the synthesis of triacylglycerols, phospholipids and cholesterol esters [145], It is in these reactions that the peculiar reactivity of thioesters is of such significance. Many hydrolases, and mainly mitochondrial thiolester hydrolases (EC 3.1.2), are able to cleave thioesters. In addition, cholinesterases and carboxylesterases show some activity, but this is not a constant property of these enzymes since, for example, carboxylesterases from human monocytes were found to be inactive toward some endogenous thioesters [35] [146], In contrast, allococaine benzoyl thioester was found to be a good substrate of pig liver esterase, human and mouse butyrylcholinesterase, and mouse acetylcholinesterase [147],... 

S. J. Gatley, Activities of the Enantiomers of Cocaine and Some Related Compounds as Substrates and Inhibitors of Plasma Butyrylcholinesterase , Biochem. Pharmacol. 1991, 41, 1249-1254. 

The cholinesterases, acetylcholinesterase and butyrylcholinesterase, are serine hydrolase enzymes. The biological role of acetylcholinesterase (AChE, EC 3.1.1.7) is to hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline (Scheme 6.1). This plays a role in impulse termination of transmissions at cholinergic synapses within the nervous system (Fig. 6.7) [12,13]. Butyrylcholinesterase (BChE, EC 3.1.1.8), on the other hand, has yet not been ascribed a function. It tolerates a large variety of esters and is more active with butyryl and propio-nyl choline than with acetyl choline [14]. Structure-activity relationship studies have shown that different steric restrictions in the acyl pockets of AChE and BChE cause the difference in their specificity with respect to the acyl moiety of the substrate [15]. AChE hydrolyzes ACh at a very high rate. The maximal rate for hydrolysis of ACh and its thio analog acetyl-thiocholine are around 10 M s , approaching the diffusion-controlled limit [16]. 

Acetylcholine is the optimal substrate for acetylcholinesterase, whereas butyrylcholine is the optimal substrate for butyrylcholinesterase. 80,104 Acetylcholinesterase does not hydrolyze butyrylcholine very efficiently-... 

Further search for inverse substrates other than /7-amidinophenyl esters has been carried out and it has been found that esters derived from p-amin omcthylphcnol and /7-guanidinophenol were also eligible as a substrate of trypsin and trypsin-like enzymes 75 86). We have also found that trimethylaminobutanoic acid p-nitrophenyl ester is an inverse substrate for butyrylcholinesterase 87-88(. Application of the inverse concept to thiol enzymes was also successful p-amidinophenyl esters were found to be substrates for clostripain 74), a thiol enzyme with trypsin-like specificity. Although the design of inverse-type substrates seems not always possible for a variety of hydrolytic enzymes, this new concept could provide potential means for certain enzymes to both fundamental study and application. 

Substrates acetylthiocholine for ChE, butyrylthiocholine for BuChE, and acetylthiocholine and 0.1 mM iso-OMPA. Abbreviations AChE, acetylcholinesterase BuChE, butyrylcholinesterase ChE, cholinesterase iso-OMPA, tetraisopropylpyrophosphoramide Source Trainaand Serpietri (1984). 

Acyl Cholinesterases. Acetylcholinesterase (AChE EC 3.1.1.7 CAS 9000-81-1) is the serine esterase which catalyzes the hydrolysis of acetylcholine and possesses an esteratic site, and which is responsible for unspecific hydrolyses of several substrates. Also, butyrylcholinesterase (EC 3.1.1.8 CAS 9001-08-5) has been sometimes used for asymmetric hydrolysis of esters. Acetylcholinesterase has been used for... 

There are two types of cholincstcra.scs in humans. ACliEri butyrylcholinesterase (BuChE). The cholincsteRLscsdiftar their location in the body and their substrate spedfsr-... 

ANTICHOLINESTERASES are agents that inhibit cholinesterases, enzymes that fall into two main families -acetylcholinesterases (AChE) and butyrylcholinesterases (BChE). These enzymes are of related molecular structures but have different distributions, genes and substrate preferences. The enzymes have globular catalytic subunits that are the soluble form of the esterases (as in plasma or CSF), or they can be attached via long collagen tails to the cell membrane. 

Gatley, S.J. Activities of the enantiomers of cocaine and some related compounds as substrates and inhibitors of plasma butyrylcholinesterase. Biochem Pharmacol 41 1249-1254, 1991. 

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. 

Nicolet Y. et al. Crystal stmcture of human butyrylcholinesterase and of its complexes with substrate and products, J. Biol. Chem., 278, 41141, 2003. 

Activation at high substrate concentrations not only explains the failure of butyrylcholinesterase to follow simple Michaelis-Menton kinetics, but also explains the enigma of substrate inhibition of the enzyme using either benzoylcholine (A21, T7) or acetyl- or butyryl-salicylcholine as substrates. The proposal made by Hastings is analogous to that of Myers (M24, M25) for the inhibition of acetylcholinesterase by excess substrate, in this case acetylcholine. 

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). 

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). 

The effects of inorganic salts on plasma cholinesterase (E16) are largely contradictory. Fruentova (F9) reported that divalent cations are more effective inhibitors of horse serum cholinesterase than are monovalent ions, whereas divalent ions are frequently reported to have a marked activating effect (H38, T8, VI). Lithium and sodium nitrates have been shown by in vitro studies of the reaction of human plasma cholinesterase with benzoylcholine to have identical inhibition profiles (W21), while sodium and potassium chlorides had very similar inhibitory actions on the hydrolysis of acetylcholine by human plasma (H47). Silver nitrate, copper sulfate, and mercuric chloride are powerful inhibitors of F. polycolor butyrylcholinesterase (N2). Cohen and Oosterbaum (C12) concluded that activation by cations occurring at the usual substrate concentration is highly dependent on the experimental conditions. This supposition is very relevant to the somewhat random choice of buffers and substrates in the work reported above. 

C7. Ghiu, Y. G., Tripathi, R. K., and O Brien, R. D., Differences in reactivity of four butyrylcholinesterase isozymes towards substrate and inhibitors. Biochem. Biophys. Res. Commun. 46, 35-42 (1972). 

A common ancestral gene may have encoded CES, acetylcholinesterase (AGhE), and thyroglobulin. Acetylcholinesterase and butyrylcholinesterase (BChE), like the CES enzymes, are members of the serine hydrolase superfamily. Cocaine (Table 8.1) is metabolized by hCEl, hCE2, and serum BChE. Though, as in the case of cocaine, there can be substrate overlap, each of these enzymes can also hydrolyze different substrates. There is noticeable sequence homology between them, particularly in the N-terminal half that includes the active serine site. 

There are two main enzymes of interest. Acetylcholinesterase (AChE EC 3.1.1.7) has an affinity for the substrate acetylcholine and it is found in the erythrocytes and nervous tissue. The enzyme is sometimes referred to as true cholinesterase, and it exists in differing polymorphic forms (Skau 1985). Butyrylcholinesterase (BuChE, acylcholine acylhydrolase, EC 3.1.1.8)—also known as pseudocholinesterase or nonspecific cholinesterase— has affinities for the substrates butyrylcholine and/or pro-pionylcholine, which are dependent on the animal species (Myers 1953 Ecobichon and Comeau 1973 Scarsella et al. 1979 Unakami et al. 1987 Evans 1990 Matthew and Chapin 1990 Woodard et al. 1994). 

Waiskopf, N., Shweky, I., Lieberman, L, et al, 2011. Quantum dot labeling of butyrylcholinesterase maintains substrate and inhibitor interactions and cell adherence features. ACS Chem. Neurosci. 2 (3), 141-150. 

The activities of two enzymes have been used as biomarkers of effects for OPs, namely acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase, sometimes known as pseudocholinesterase (EC 3.1.1.8). The structure and function of these enzymes has been reviewed. " In humans the former is present in red blood cells and the latter in plasma, but such distribution is not true of all species. In dogs, both enzymes are present in plasma with a ratio of butyrylcholinesterase to acetylcholinesterase of 7 1, while in the rat, plasma cholinesterase activity comprises more acetylcholinesterase with a butyrylcholinesterase to acetylcholinesterase activity of 1 3 in males and 2 1 in females in neither blood compartment are the functions of the enzymes fully understood.Because of the possibility of confusion, the terms plasma cholinesterase and erythrocyte cholinesterase as synonyms for butyrylcholinesterase and acetylcholinesterase are to be deprecated, especially when used of enzymes in animals where serious confusion may result. It is often necessary to look in detail at animal studies to see what activity has been measured in each matrix. In particular, it is necessary to look at the substrate(s) used in the assay together with any inhibitors used. Methods for measuring acetylcholinesterase have been reviewed and acetylcholinesterase and butyrylcholinesterase activities can be measured separately. In almost all cases it is the enzyme activity, rather than protein concentration, that is measured and many of the procedures used are variants of the Ellman method. Correct storage of blood samples is important as reactivation of inhibited enzymes ex vivo can occur. 

Figure 8.10 Schematic representation cf an automated system for the determination of pesticides using a butyrylcholinesterase (BuChE) electrode (R - r erence electrode E - enzyme electrode BuChCl -butyrylcholine chloride (substrate) PAM - reactivator).

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