Acetylcholinesterase, suicide inhibitors

Irreversible inhibitors combine or destroy a functional group on the enzyme so that it is no longer active. They often act by covalently modifying the enzyme. Thus a new enzyme needs to be synthesized. Examples of irreversible inhibitors include acetylsal-icyclic acid, which irreversibly inhibits cyclooxygenase in prostaglandin synthesis. Organophosphates (e.g., malathion, 8.10) irreversibly inhibit acetylcholinesterase. Suicide inhibitors (mechanism-based inactivators) are a special class of irreversible inhibitors. They are relatively unreactive until they bind to the active site of the enzyme, and then they inactivate the enzyme. 

Metabolites that are less reactive than suicide inhibitors may impact more distant enzymes, within the same cell, adjacent cells, or even in other tissues and organs, far removed from the original site of primary metabolism. For example, organopho-sphates (OPs), an ingredient in many pesticides, are metabolized by hepatic CYPs to intermediates, which, when transported to the nervous system, inhibit esterases that are critical for neural function. Acetylcholinesterase (AChE) catalyzes the hydrolysis of the ester bond in the neurotransmitter, acetylcholine, allowing choline to be recycled by the presynaptic neurons. If AChE is not effectively hydrolyzed by AChE in this manner, it builds up in the synapse and causes hyperexcitation of the postsynaptic receptors. The metabolites of certain insecticides, such as the phos-phorothionates (e.g., parathion and malathion) inhibit AChE-mediated hydrolysis. Phosphorothionates contain a sulfur atom that is double-bonded to the central phosphorus. However, in a CYP-catalyzed desulfuration reaction, the S atom is... 

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... 

Clavulanic acid is a mechanism-based irreversible inhibitor and could be classed as a suicide substrate (Chapter 4). The drug fits the active site of (3-lactamase and the 13-lactam ring is opened by a serine residue in the same manner as penicillin. However, the acyl-enzyme intermediate then reacts further with another enzymic nucleophilic group (possibly NH2) to bind the drug irreversibly to the enzyme (Fig. 10.54). The mechanism requires the loss or gain of protons at various stages and an amino acid such as histidine present in the active site would be capable of acting as a proton donor/acceptor (compare the mechanism of acetylcholinesterase in Chapter 11). 

Other types of inhibitors may not be so tolerable. Organophosphorus compounds, used in nerve gases and weed killers (e.g., parathion), form a covalent irreversible bond with the active serine and permanently inactivate acetylcholinesterase. This is a type of suicide inhibition because the inhibitor reacts with the enzyme much like a substrate, but becomes blocked in the intermediate state where the enzyme-phosphoryl bond is stable, in contrast to the hydrolyzable enzyme-acetyl bond. These compounds are life-threatening. 

Five novel carbonates/ designed as suicide (mechanism-based) inhibitors of acetylcholinesterase, were synthesized and evaluated against the enzyme in vitro and screened for insecticidal activity. The design strategy of inhibition was based on the isosteric relationship of carbonates to the ester of the natural substrate acetylcholine, and on the release of electrophilic quinone methides or alpha-chloroketones at the active site after enzymatic carbonate hydrolysis. Most coirpounds were inhibitory in vitro, with good specificity for acetylcholinesterase. Some showed modest insecticidal activity. Results of kinetic studies on one analog were consistent with mechanism-based inhibition. 

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