Acetylcholinesterase substrates

Enzyme-Catalyzed Reactions Enzymes are highly specific catalysts for biochemical reactions, with each enzyme showing a selectivity for a single reactant, or substrate. For example, acetylcholinesterase is an enzyme that catalyzes the decomposition of the neurotransmitter acetylcholine to choline and acetic acid. Many enzyme-substrate reactions follow a simple mechanism consisting of the initial formation of an enzyme-substrate complex, ES, which subsequently decomposes to form product, releasing the enzyme to react again. 

So far, many kinds of nucleophiles active for hydrolysis such as imidazolyl-, amino-, pyridino-, carboxyl- and thiol-groups, have been used for preparation of hydrolase models. Overberger et al.108,1091 prepared copolymers of vinylimidazole and acrylic acid 60 (PVIm AA), by which the cationic substrate, 61 (ANTI), was hydrolyzed. This kind of copolymer is considered to be a model of acetylcholinesterase. With ANTI, the rate of the copolymer catalysis was higher than that of imidazole itself in the higher values of pH, as is seen in Table 9. In this work, important contributions of the electrostatic interactions are clear. The activity of the copolymer was not as high with the negatively charged and neutral substrates. 

Acetylcholine serves as a neurotransmitter. Removal of acetylcholine within the time limits of the synaptic transmission is accomplished by acetylcholinesterase (AChE). The time required for hydrolysis of acetylcholine at the neuromuscular junction is less than a millisecond (turnover time is 150 ps) such that one molecule of AChE can hydrolyze 6 105 acetylcholine molecules per minute. The Km of AChE for acetylcholine is approximately 50-100 pM. AChE is one of the most efficient enzymes known. It works at a rate close to catalytic perfection where substrate diffusion becomes rate limiting. AChE is expressed in cholinergic neurons and muscle cells where it is found attached to the outer surface of the cell membrane. 

The rhodium complex of the (R,R)-counter-enantiomer of (S,S)-BisP achieved a high level of ee (97%) in the asymmetric hydrogenation of 3-methoxy-substituted substrate (S)-122 (Scheme 25), which constitutes a precursor to the acetylcholinesterase inhibitor SDZ-ENA-713 (123). 

Hussein, A.S., Smith, A.M., Chacon, M.R. and Selkirk, M.E. (2000) A second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis determinants of substrate specificity. European Journal of Biochemistry 267, 2276-2282. 

Ordendich, A., Barak, D., Kronman, C., Flashner, Y., Leitner, M., Segall, Y., Ariel, N., Cohen, S., Velan, B. and Shafferman, A. (1993) Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Journal of Biological Chemistry 268, 17083—17095. 

Ripoll, D.R., Faerman, C.H., Axelson, P.H., Silman, I. and Sussman, J.L. (1993) An electrostatic mechanism for substrate guidance down the aromatic gorge of acetylcholinesterase. Proceedings of the National Academy of Sciences USA 90, 5128-5132. 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

Observations The preliminary treatment of the cholinesterase-containing material with allelochemical (or other compound, e.g. active oxygen species, ozone free radicals and peroxides, formed in allelopathic relations) is for 30 min, then a substrate acetylcholinesterase is added to the reaction medium and final reaction of hydrolysis is for 1 h. 

C. la Rosa, F. Pariente, L. Hernandez, and E. Lorenzo, Determination of organophosphorus and car-bamic pesticides with an acetylcholinesterase amperometric biosensor using 4-aminophenyl acetate as substrate. Anal. Chim. Acta 295, 273-282 (1993). 

Cholinesterases are widely distributed throughout the body in both neuronal and non-neuronal tissues. Based largely on substrate specificity, the cholinesterases are subdivided into the acetylcholinesterases (AChEs) (EC... 

Cholinesterases are subdivided into acetylcholinesterase and cholinesterase, one with a narrow, the other with broad substrate specificity [109-112], Both enzymes exist in multiple molecular forms distinguishable by their subunits association (Fig. 2.4). The hydrodynamic properties of these associations have allowed globular (G) and asymmetric (A) forms to be distinguished. The G forms can be hydrophilic (water-soluble, and excreted into body fluids) or amphiphilic (membrane-bound). The homomeric class exists... 

Enol esters are distinct from other esters not because of a particular stability or lability toward hydrolases, but due to their hydrolysis releasing a ghost alcohol (an enol), which may immediately tautomerize to the corresponding aldehyde or ketone. A well-studied example is that of vinyl acetate (CH3-C0-0-CH=CH2), a xenobiotic of great industrial importance that, upon hydrolysis, liberates acetic acid (CH3-CO-OH) and acetaldehyde (CH3-CHO), the stable tautomer of vinyl alcohol [25], The results of two studies are compiled in Table 7.1, and demonstrate that vinyl acetate is a very good substrate of carboxylesterase (EC 3.1.1.1) but not of acetylcholinesterase (EC 3.1.1.7) or cholinesterase (EC 3.1.1.8). The presence of carboxylesterase in rat plasma but not in human plasma explains the difference between these two preparations, although the different experimental conditions in the two studies make further interpretation difficult. 

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],... 

Phosphinates are a class of organophosphorus compounds, the metabolism of which has received less attention than that of phosphates (see above) or phosphorothioates and P-halidc compounds (see below). Many phosphinates are rapid but transient inhibitors of acetylcholinesterase and carboxyl-esterases. And like organophosphates and phosphonates, phosphinates are substrates of arylesterases (EC 3.1.1.2). This is exemplified by 4-nitrophen-yl ethyl(phenyl)phosphinate (9.62), whose (-)-enantiomer was hydrolyzed by rabbit serum arylesterase almost 10 times faster than the (+)-enantiomer [133],... 

HPMA copolymers are water-soluble biocompatible polymers, widely used in anticancer drug delivery (reviewed in Reference [22]). HPMA copolymers containing reactive groups at side-chain termini were previously used for the modification of trypsin [23], chymotrypsin [23,24], and acetylcholinesterase [25]. The modification dramatically increased the acetylcholinesterase survival in the blood stream of mice and the thermostability of modified enzymes when compared to the native proteins. However, the modification involved multipoint attachment of the copolymers to the substrates, which may cause crosslinking. To modify proteins or biomedical surfaces by one point attachment, semitelechelic polymers should be used. 

Most enzymes bind their substrates in a non-covalent manner but, for those that do bind covalently, the intermediate must be less stable than either substrate or product. Many of the enzymes that involve covalent catalysis are hydrolytic enzymes these include proteases, lipases, phosphatases and also acetylcholinesterase. Some of these enzymes possess a serine residue in the active site, which reacts with the substrate to form an acylenzyme intermediate that is attacked by water to complete the hydrolysis (Fignre 3.3). 

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]. 

In this drug class, only sucdnylcholine (succinyldicholine, suxamethonium, AJ is of clinical importance. Structurally, it can be described as a double ACh molecule. Like ACh, succinylcholine acts as agonist at endplate nicotinic cholino-ceptors, yet it produces muscle relaxation. Unlike ACh, it is not hydrolyzed by acetylcholinesterase. However, it is a substrate of nonspecific plasma cholinesterase (serum cholinesterase, p. 100). 

A reaction whose rate is limited (or controlled) only by the speed with which reactants diffuse to each other. For a ligand binding to a protein, the bimolecular rate constant for diffusion-limited association is around 10 M s. The enzyme acetylcholinesterase has an apparent on-rate constant of 1.6 x 10 M s with its natural cationic substrate acetylcholine, and the on-rate constant of about 6 X 10 with acetylselenoylcholine and about... 

The answer is D. Organophosphates react with the active site serine residue of hydrolases such as acetylcholinesterase and form a stable phosphoester modification of that serine that inactivates the enzyme toward substrate. Inhibition of acetylcholinesterase causes overstimulation of the end organs regulated by those nerves. The symptoms manifested by this patient reflect such neurologic effects resulting from the inhalation or skin absorption of the pesticide diazinon. 

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