Acetylcholinesterase inhibition irreversible

In cases where the mode of action is the strong or irreversible inhibition of an enzyme system, the assay may measure the extent of inhibition of this enzyme. This may be accomplished by first measuring the activity of the inhibited enzyme and then making comparison with the uninhibited enzyme. This practice is followed when studying acetylcholinesterase inhibition by organophosphates (OP). Acetylcholinesterase activity is measured in a sample of tissue of brain from an animal that has been exposed to an OP. Activity is measured in the same way in tissue samples from untreated controls of the same species, sex, age, etc. Comparison is then made between the two activity measurements, and the percentage inhibition is estimated. 

A third approach to protection against excessive acetylcholinesterase inhibition is pretreatment with reversible enzyme inhibitors to prevent binding of the irreversible organophosphate inhibitor. This prophylaxis can be achieved with pyridostigmine but is reserved for situations in which possibly lethal poisoning is anticipated, eg, chemical warfare (see Chapter 7). Simultaneous use of atropine is required to control muscarinic excess. 

Acetylcholinesterase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. As the inhibition is irreversible and stoichiometric, it is possible to calculate the toxin concentration by the percentage of the enzyme that has not been inhibited and remains active. The use of several enzyme concentrations and the... 

The previous discussion of amino acid catabolic disorders indicates that catabolic processes are just as important for the proper functioning of cells and organisms as are anabolic processes. This is no less true for molecules that act as neurotransmitters. To maintain precise information transfer, neurotransmitters are usually quickly degraded or removed from the synaptic cleft. An extreme example of enzyme inhibition illustrates the importance of neurotransmitter degradation. Recall that acetylcholine is the neurotransmitter that initiates muscle contraction. Shortly afterwards, the action of acetylcholine is terminated by the enzyme acetylcholinesterase. (Acetylcholine must be destroyed rapidly so that muscle can relax before the next contraction.) Acetylcholinesterase is a serine esterase that hydrolyzes acetylcholine to acetate and choline. Serine esterases have catalytic mechanisms similar to those of the serine proteases (Section 6.4). Both types of enzymes are irreversibly inhibited by DFP (diisopropylfluorophosphate). Exposure to DFP causes muscle paralysis because acetylcholinesterase is irreversibly inhibited. With each nerve impulse, more acetylcholine molecules enter the neuromuscular synaptic cleft. The accumulating acetylcholine molecules repetitively bind to acetylcholine receptors. The overstimulated muscle cells soon become paralyzed (nonfunctional). Affected individuals suffocate because of paralyzed respiratory muscles. 

Diethyl 0-(3-methyl-5-pyrazolyl) phosphate (722) and 0,0-diethyl 0-(3-methyl-5-pyrazolyl) phosphorothioate (723) were prepared in 1956 by Geigy and they act, as do all organophosphates in both insects and mammals, by irreversible inhibition of acetylcholinesterase in the cholinergic synapses. Interaction of acetylcholine with the postsyn-aptic receptor is therefore greatly potentiated. 0-Ethyl-5-n-propyl-0-(l-substituted pyrazol-4-yl)(thiono)thiolphosphoric acid esters have been patented as pesticides (82USP4315008). 

There is a second type of cholinesterase called butyrylcholinesterase, pseudocholinesterase, or cholinesterase. This enzyme is present in some nonneural cells in the central and peripheral nervous systems as well as in plasma and serum, the liver, and other organs. Its physiologic function is not known, but is hypothesized to be the hydrolysis of esters ingested from plants (Lefkowitz et al. 1996). Plasma cholinesterases are also inhibited by organophosphate compounds through irreversible binding this binding can act as a detoxification mechanism as it affords some protection to acetylcholinesterase in the nervous system (Parkinson 1996 Taylor 1996). 

Figure 3. Plot of V against total enzyme [ET] showing the irreversible inhibition of el tric eel acetylcholinesterase (AChE) by ANTX-A(S). The enzymes were incubated with 0.32 fig/mL ANTX-A(S) for 1.0 min and acetylthiocholine (final concentrations 2.5, 4.7, 6.3, and 7.8 X 10 M) was added. V was determined from the double reciprocal plots (not shown). Key (o) control ( ) ANTX-A(S). (Reproduced with permission from Ref. 42. Copyright 1987 Pergamon Press)...

ACh is metabolised extraneuronally by the enzyme acetylcholinesterase, to reform precursor choline and acetate. Blocking its activity with various anticholinesterases has been widely investigated and some improvement in memory noted. Such studies have invariably used reversible inhibition because of the toxicity associated with long-term irreversible inhibition of the enzyme. Physostigmine was the pilot drug. It is known to improve memory in animals and some small effects have been seen in humans (reduces number of mistakes in word-recall tests rather than number of words recalled), but it really needs to be given intravenously and has a very short half-life (30 min). 

Irreversible inhibition in an organism usually results in a toxic effect. Examples of this type of inhibitor are the organophosphorus compounds that interfere with acetylcholinesterase (see Box 7.26). The organophosphorus derivative reacts with the enzyme in the normal way, but the phosphory-lated intermediate produced is resistant to normal hydrolysis and is not released from the enzyme. 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

Obidoxime is an antidote used to treat poisoning with insecticides of the organophosphate type (p. 102). Phosphorylation of acetylcholinesterase causes an irreversible inhibition of ace-Ltillmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of license. 

Bronchoconstriction and secretion, muscular weakness or paralysis, CNS depression, including respiratory centers Inhibition of acetylcholinesterase (reversible or irreversible)... 

Acetylcholinesterase is subject to substrate Inhibition at high concentrations, but Mlchaells kinetics are observed at lower concentrations, because the substrate constant and the Mlchaells constant differ by a factor of 100. Turnover numbers run about 2-9 x 10 min l, and (Mlchaells constant) values are about 0.2 mM.76,116 Whatever the source, the enzyme is subject to inhibition by the same reversible and irreversible inhibitors. Most of the kinetic work has been done with the saline-extracted 11S enzyme from electric eel and the detergent-extracted 6S enzyme from erythrocytes. The former Is a tetramer derived from the native enzyme by the action of proteases the latter is a dimer. 

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. 

It is important to know that the inhibition of acetylcholinesterase by OPs is through an attack on the relatively positive phosphorus atom by the hydroxyl group of a serine residue at the enzyme s site of action. Electron withdrawing substitutions within the OP tend to make the phosphorus more positive and, therefore, more reactive. Unfortunately, this type of substitution also makes the compound less stable hydrolytically. The discovery and development of OP insecticides has always been a balance between activity against the enzyme of the insect, selectivity in comparison with mammalian systems and stability within the insect. The binding of OPs to acetylcholinesterase is often irreversible. Typical OP insecticides are shown in Figure 3.3. 

Irreversible inhibition - This type of inhibition involves the stable combination of an enzyme with some species so that the only way to have enzyme activity is to produce more enzyme. Specific examples of irreversible inhibition will be covered in Part II of the course in the section on the nervous system. Organophosphate and carbamate insecticides are the prime examples of such inhibitors when they combine with the enzyme acetylcholinesterase. 

The mode of action, which is now well authenticated and understood, involves the irreversible inhibition of the enzyme acetylcholinesterase, which is essential to nervous conduction in insects, by phosphorylation of a hydroxy group at the active site. The detailed mechanisms have been reviewed by O Brien (B-67MI10700, B-76MI10701). 

Anatoxin-a(s) is a phosphate ester of a cyclic iV-hydroxyguanidine (Fig. 16.2) [5]. It is the only natural organophosphate known and, as the synthetic parathion and malathion, irreversibly inhibits acetylcholinesterase. When this enzyme is inhibited, acetylcholine is no longer hydrolysed, the postsynaptic membrane cannot be repolarised, the nerve influx is blocked and the muscle cannot be contracted. 

Anatoxin-a(s) was first detected by the mouse bioassay. However, the technique most commonly used for its detection is HPLC, coupled to MS detection. The irreversible inhibitory power of this toxin towards acetylcholinesterase has been described [60] and the corresponding colorimetric inhibition assay has also been developed [61-63]. To date, no antibodies towards anatoxin-a(s) have been produced. 

The developed biosensor was applied to the analysis of cyanobacterial bloom samples from freshwater lakes of Spain, Greece, France, Scotland and Denmark. Two samples from Scotland and one from Denmark irreversibly inhibit the acetylcholinesterase. The estimated concentrations were between 1.5 and 30nmol/g of dry weight, values extremely high when compared to the intraperitoneal 50% lethal dose of anatoxin-a(s) in mice (121 nmol/kg). 

Diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF), both organophosphorus inhibitors, are potent irreversible inhibitors of serine proteases. However, because of their additional inhibition of acetylcholinesterase these compounds are highly toxic [26]. Another toxic but potent trypsin inhibitor is (4-aminophenyl)-methanesulfonyl... 

Ser residue in the active site of the enzyme acetylcholinesterase, irreversibly inhibiting the enzyme and preventing the transmission of nerve impulses (Fig. la). Iodoacetamide modifies Cys residues and hence may be used as a diagnostic tool in determining whether one or more Cys residues are required for enzyme activity (Fig. lb). The antibiotic penicillin irreversibly inhibits the glycopeptide transpeptidase enzyme that forms the cross-links in the bacterial cell wall by covalently attaching to a Ser residue in the active site of the enzyme (see Topic Al). 

Among the irreversible inhibitors are organophosphorus compounds, which inhibit the enzyme acetylcholinesterase and similar enzymes. Organophosphorous compounds include nerve gases (such as sarin), that work on the human nervous system, and insecticides like malathion. 

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