Acetylcholinesterase substrate specificity

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. 

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... 

The frequent occurrence of sialylated enzymes, or even of multiple forms, which are sometimes tissue-dependent, with a varying number of sialyl residues as, for example, in y-glutamyltranspeptida.se (EC 2.3.2.2),456,457 is not yet fully understood. Although the activity of most of these enzymes is not influenced by removal of sialic acid,454 the activity of monoamine oxidase A (EC 1.4.3.4) of outer mitochondrial membranes of rat liver has been shown to be destroyed by treatment with sialidase438 the substrate specificity of acetylcholinesterase (EC 3.1.1.7) is altered,459 the kinetic properties of human acid and alkaline phosphatases (EC 3.1.3.1 and 3.1.3.2) are changed, and the stability of a-D-galactosidase (EC 3.2.1.22) is drastically lowered.415 In these cases, an influence of sialyl residues on the conformation of the enzyme is assumed, but awaits firm evidence. 

Harel, M., Quinn, D.M., Nair, H.K., Silman, I., Sussman, J.L. The X-ray Structure of a Transition State Analog Complex Reveals the Molecular Origins of the Catalytic Power and Substrate Specificity of Acetylcholinesterase./. Am. Chem. Soc. 1996, 118, 2340-2346. 

M. Harel, D. M. Quinn, H. K. Nair, I. Silman and J. L. Sussman, The X-ray structure of a transition state analog complex reveals the molecular origins of the catalytic power and substrate specificity of acetylcholinesterase, /. Am. Chem. Soc., 1996,118, 2340-2346 (pdb lamn). 

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. 

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 contrast to acetylcholinesterase, which is selective for acetylcholine, butyryl-cholinesterase tolerates a wider variety of esters and is more active with butyryl-and propionylcholines than acetylcholine [7]. Structure-activity relationship studies have shown that different steric restrictions in the acyl pockets of AChE and BChE cause the difference in specificity to the acyl moiety of the substrate [6]. 

Most irreversible enzyme inhibitors combine covalently with functional groups at the active sites of enzymes. These inhibitors are usually chemically reactive, and many of them show some specificity in terms of the amino acid groups which they react with. Diisopropyl fluorophosphate (DFP), for example, forms a covalent adduct with active site serine residues, such as in the serine proteases, and in acetylcholinesterase, which explains its toxic effect on animals. Irreversible enzyme inhibition can be used to identify important active site residues. A special case of irreversible enzyme inhibition is the effect of suicide inhibitors, which are generally chemically unreactive compounds that resemble the substrate of the target enzyme and bind at the active site. The process of enzyme turnover begins, but the inhibitor is so... 

The effect of irreversible inhibition of acetylcholinesterase has been used in dendrimer-based electrochemical biosensors for environmental applications. Acetylcholinesterase is a very efficient protein catalyst for the hydrolysis of its physiological substrate acetylcholine. Organophosphorus and carbamic pesticides, heavy metals and detergents exert strong specific... 

Acetylcholinesterase (AChE) (also termed true cholinesterase ) is found in the synaptic cleft of cholinergic synapses, and is of undoubted importance in regulation of neurotransmission by rapid hydrolysis of released endogenous acetylcholine (ACh). AChE is also found in erythrocytes and in the CSF, and can be present in soluble form in cholinergic nerve terminals, but its function at these sites is not clear, AChE is specific for substrates that include acetylcholine and the agents methacholine and acetylthiocholine. but it has little activity with other esters. It has a maximum turnover rate at very low concentrations of AChE (and is inhibited by high concentrations). 

Elucidation of the molecular nature of another cholinergic receptor system, the enzyme acetylcholinesterase. is also being attempted through the use of specific, irreversible inhibitors. Belleau and Tanl have recently demonstrated that N,N-dlmethyl-2-chloro-2 phenylethylamine inactivates erythrocyte acetylcholinesterase when acetylcholine is the substrate.. Other workers have provided evidence that the action of this compound involves alkylation at or near the anionic site of the enzyme, rather then at the esteratlc site. Future studies of this system may well be conducted with pure, crystalline enzyme, if a recent report" of the crystallization of acetylcholinesterase from eel electric tissue can be confirmed. 

Fig 5-2. This schematic ribbon diagram shows the structure of Torpedo californica acetylcholinesterase. The diagram is color-coded green the 537-amino acid polypeptide of the enzyme monomer pink the 14 aromatic residues that line the deep aromatic gorge leading to the active site and gold and blue a model of the natural substrate for acetylcholinesterase, the neurotransmitter acetylcholine, docked in the active site. Reprinted with permission from Sussman JL, Silman I. Acetylcholinesterase Structure and use as a model for specific cation-protein interactions. Curr Opin Struct Biol. 1992 2 724. 

Because the ACh receptor does not hydrolyse acetylcholine, the esteratic site of acetylcholinesterase (see Fig. 12.3) must be absent. Other fundamental differences in the two sites are indicated by the following (a) dimethylbutyl acetate 12.66) is a good substrate for the enzyme, but barely activates the receptor which requires a basic group for marked activity (b) muscarine is not a substrate for the enzyme and yet it is a powerful agonist for the muscarinic receptor (c) di-isopropyl phosphorofluoridate (isoflurophate) 13.26) binds to the active site of AChase but not to an ACh receptor (d) acetyl-bungarotoxin specifically binds to the ACh nicotinic receptor but not to the enzyme. 

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