Acetylcholinesterase catalytic site

The ligand binding or catalytic sites are the most relevant parts of a protein domain for the development of small molecules as modulators of protein function. There is evidence that proteins with conserved folds often also have their functional sites on the same topological location. In some cases a remarkable conservatism in functional sites can be observed. This is true for the example described later in this review on similarity of Cdc25A phosphatase, acetylcholinesterase (AChE) and 1 Ifl-hydroxysteroid dehydrogenases (1 l HSD) (Fig. 9). Nevertheless, it should be stressed that the correlation patterns of amino acid sequence, protein fold and protein function remain a matter of debate. Moreover, a vast number of specific functions can be carried out by the limited number of protein domains due to the high amino acid diversity of proteins with similar folds. " ... 

Oxazocine 119 is a synthetic derivative of galanthamine 162. The latter is a tertiary alkaloid, isolated from amaryllidaceae, which is a central acting competitive and reversible inhibitor of acetylcholinesterase that enhances cognitive functions in Alzheimer s patients. However, oxazocine 119 showed a decreased potency as an acetylcholinesterase inhibitor and a marked selectivity with respect to butyrylcholinesterase, probably because butyrylcholinesterase accommodates steric bulk around the catalytic site, better than acetylcholinesterase <2003BML2389>. 

Pyridostigmine is a carbamate that is used in the treatment of myasthenia gravis, a neuromuscular disease. It can also be used as a method of protection against nerve agent poisoning. Carbamates can occupy the catalytic site of acetylcholinesterase (AchE), which temporarily prevents phosphorylation. 

Replacement of one or more of the hydrogen atoms of the ethylene bridge with alkyl groups produces marked changes in potency and activity. Acetyl /3-methylcholine (31) is equipotent to acetylcholine as a muscarinic agonist, but it has a much weaker nicotinic action (74). A factor in the observed potency of acetyl ]8-methylcholine is its slower rate of hydrolysis by acetylcholinesterase because of poor affinity of the compound for the enzyme s catalytic site (81) and its extremely high resistance to hydrolysis by nonspecific serum cholinesterases. 

Remember too that it was the research of insecticides that led to the discovery of the organophosphorus acetylcholinesterase inhibitors by Schrader at the Bayer laboratory. The stndy of their mechanism of action has shown that they act by acylation of a serine hydroxyl in the catalytic site of the enzyme. This was one of the first examples describing a molecular mechanism for an enzymatic inhibition. 

Shafferman, A., Ordentlich, A., Barak, D., et al., 1996. Aging of phosphylated human acetylcholinesterase catalytic processes mediated by aromatic and polar residues of the active site. Biochem. J. 318, 833-840. 

FIGURE 5.46 Interaction of the serine hydroxyl residue in the catalytically active site of acetylcholinesterase enzyme with esters of organophosphates or carbamates. The interaction leads to binding of the chemical with the enzyme, inhibition of the enzyme, inhibition of acetylcholine hydrolysis, and thus accumulation of acetylcholine in the synapses. 

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. 

All of these esterases appear to act by mechanisms closely related to those of proteases. Acetylcholinesterase contains an active site serine that reacts with organophosphorus compounds (Box 12-E) and is part of an Asp-His-Ser catalytic triad which lies in a deep "gorge" as well as an oxyanion hole.637 A surprise is the absence of an essential carboxylate group that might bind the positively charged trimethylammonium... 

Vigny, M, Bon, S, Massoulie, J, and Leterner, F (1978) Active site, catalytic efficiency of acetylcholinesterase molecular forms in electrophorus, torpedo, rat and chicken Eur J Biochem. 85, 317—323. 

Figure 9.17. Organophosphate inhibitors of acetylcholinesterase. a The catalytic mechanism, shown here for diiso-propylfluorophosphate(DFP).b Stmcturesof soman and tabun. Like DFP, these were developed during world war II as nerve gases , c Stractures of the insecticides parathion and malathion, and of paraoxon, which is the achve metabolite of parathion. (Malathion likewise requires conversion to malaoxon.) The arrow above the malathione stmcture indicates the esterase cleavage sites in its leaving group esterase cleavage occurs in human plasma and renders the molecule non-toxic.

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). 

In a discovery project that is reminiscent of the discovery of captopril, scientists at Takeda created a hypothetical structure for the active site of acetylcholinesterase, based on SAR from previous biochemical and medicinal chemical work (141). The model consisted of (in addition to the serine protease-like catalytic machinery) an anionic binding site separating two discrete hydrophobic binding sites. This model was then used to design inhibitors of the enzyme (reviewed i n ref. 142). One set of analogs examined were based on the N-((o-phthalimidylalkyl)-iV-(a)-phenylalkyl)-amine (scaffold 66). An iterative process of testing. 

Figure 5. Variation of the protein MEP along the active sites of some enzymes. It a-chymotiypsin, 2t p-tiypsin, 3 porcine pancreatic elastase, 4 Streptomyces Griseus hydrolase, Si a-lytic protease, 6t subtilisin NOVO, 7i acetylcholinesterase, 8> lipase A, 9 lysozyme, lOi D-xyloie isomerase. Point A is at OG of die active serine in 1-8, at the bisector ofODl and OD2 of Asp-52 in 9, at Ol of the cyclic xylose m 10. Point B is atNE2 of the catalytic histidine in 1-8, in the first trisector of points A and Din 9, atNEl ofHis-S4in 10. Point C is at ND1 of the catalytic histidine in 1-8 and 10, at the second trisector of points A and D in 9. Point D is at the bisector of the carboxylate oxygens of the catalytic Asp or Glu side chains.

Acetylcholinesterase (AChE) catalyses the hydrolysis of the ester bond of acetylcholine to yield choline and acetate (Sussman et al., 1991). This is a critical reaction for the termination of impulses transmitted through cholinergic synapses. It is a highly efficient catalyst, with reaction rates approaching the diffusion limit. Its overall structure resembles the lipases with an active site gorge. Above the base of the gorge is the reactive serine to be activated by the classical (Ser-200...His-440...Glu-327) catalytic triad. 

Simple quaternary compounds such as tetra-methylammonium cation combine with the substrate cation-binding site of the catalytic surface of acetylcholinesterase and thus deny acetylcholine s access to this site. These compounds have a short duration of action due to the facile reversibility of their binding and r id renal elimination (290), and thus they have minimal therapeutic utility. Cohen and Oosterbaan (291) tabulated a comprehensive list cf tetraalkyl quaternary ammonium acetylcholinesterase inhibitors. Homologation of... 

Figure 34-18 Reactivation of phosphoryiated acetylcholinesterase by pralidoxime formation of aged phosphoryiated enzyme, which does not reactivate.The active site catalytic triad of serine, histidine, and glutamate is depicted by —OH, =NH—, and a negative charge, respectively.

Several structures of small molecule complexes with acetylcholinesterase have been solved. They reveal a binding site next to the catalytic serine preferrentially occupied by a positively charged moiety next to a hydrophobic portion. The positively charged functional groups almost superimpose in front of a trj tophan residue at the bottom of the gorge [25-27]. 

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