Irreversible inhibition, of enzymes

Describe the main features of competitive, non-competitive and irreversible inhibition of enzymes. 

In conclusion, suicide enzyme inactivators offer a powerful method for the selective irreversible inhibition of enzymes. Although this review has concentrated on pyridoxal phosphate-dependent enzymes the approach is also valid for the irreversible inhibition of other types of enzymes (6 ) and may offer a means for the rational design of therapeutically-useful substances. 

The irreversible inhibition of enzymes by suicide substrates occurs as a consequence of activation steps in which the target enzymes transform these substrates into inhibitors using their normal mechanism. 

The structural similarity between onchidal (an acetate ester) and acetylcholine suggested that the toxicity of onchidal could result from inhibition of either nicotinic acetylcholine receptors or AChE. Although onchidal (1.0 mM) did not prevent the binding of 1-a-bungarotoxin to nicotine acetylcholine receptors, it inhibited AChE in a progressive, apparently irreversible, manner. The apparent affinity of onchidal for the initial reversible binding to AChE (Kd) was approximately 300 p.M, and the apparent rate constant for the subsequent irreversible inhibition of enzyme activity (Xij ct) was approximately 0.1 min ... 

In another investigation, irreversible inhibition of enzyme activity was prevented by coincubation with reversible agents that either sterically block (edrophonium and decamethonium) or alio sterically modify (propidium) the acetylcholine site (Barnett and Rosenberry, 1977). Enzyme activity was not regenerated by incubation with oxime reactivators therefore, the mechanism of irreversible inhibition does not appear to involve acylation of the active site serine. 

Irreversible Inhibition of Enzymes 488 Box9-F Immunophilins as Rotamases... 

Enzyme inhibitors are of various types. For example, we can distinguish between irreversible and reversible inhibition. Sometimes an inhibitor reacts so strongly with the active center of an enzyme that the process cannot easily be reversed. Diisopropylfluorophosphate (DFP), for example, undergoes an irreversible reaction with the active centers of certain enzymes, with the liberation of hydrogen fluoride. One of the enzymes with which it reacts is cholinesterase, which is responsible for the functioning of the nerves. As a result DFP is a very powerful nerve gas. Poisons like potassium cyanide exert their action by the irreversible inhibition of enzymes which catalyze oxidative reactions. 

Elucidating Mechanisms for the Inhibition of Enzyme Catalysis An inhibitor interacts with an enzyme in a manner that decreases the enzyme s catalytic efficiency. Examples of inhibitors include some drugs and poisons. Irreversible inhibitors covalently bind to the enzyme s active site, producing a permanent loss in catalytic efficiency even when the inhibitor s concentration is decreased. Reversible inhibitors form noncovalent complexes with the enzyme, thereby causing a temporary de-... 

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 case similar to the slow, practically irreversible inhibition of jack bean a-D-mannosidase by swainsonine is represented by the interaction of castanospermine with isomaltase and rat-intestinal sucrase. Whereas the association constants for the formation of the enzyme-inhibitor complex were similar to those of other slow-binding glycosidase inhibitors (6.5 10 and 0.3 10 M s for sucrase and isomaltase, respectively), the dissociation constant of the enzyme-inhibitor complex was extremely low (3.6 10 s for sucrase) or could not be measured at all (isomaltase), resulting in a virtually irreversible inhibition. Danzin and Ehrhard discussed the strong binding of castanospermine in terms of the similarity of the protonated inhibitor to a D-glucosyl oxocarbenium ion transition-state, but were unable to give an explanation for the extremely slow dissociation of the enzyme-inhibitor complex. 

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). 

Gold and Linder (17) studied the esterase catalyzed hydrolysis of A-(-)-acetoxymethyl-(l-phenylethyl)nitrosamine. They found that the stereochemistry of 1-phenylethanol produced in the reaction was the same as that observed in the base catalyzed hydrolysis of the nitrosamine and also of N-(l-phenylethyl)nitrosocarbamate. These results indicated that the same diazotate was produced in all three reactions. The fact that no irreversible inhibition of the enzymatic hydrolysis of the nitrosamine was observed, while extensive irreversible inhibition was obtained with the nitrosocarba-mate, led these workers to conclude that the a-hydroxynitro-samine produced by the hydrolysis had sufficient stability to diffuse away from the active site of the enzyme. 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Sometimes CYPs can also produce reactive metabolite species that, instead of undergoing the normal detoxification pathway, can act as irreversible CYP inhibitors, thus causing toxicity. Such reactive metabolites that cause CYP inactivation are called MBI and are described in Chapter 9. Mechanism-based enzyme inhibition is associated with irreversible or quasi-irreversible loss of enzyme function, requiring synthesis of new enzymes before activity is restored. The consequences of MBI could be auto-inhibition of the clearance of the inactivator itself or prolonged inhibition of the clearance of other drugs that are cleared by the same isozyme. There may also be serious immunotoxicological consequences if a reactive intermediate is covalently bound to the enzyme. Therefore, screening of new compounds for MBI is now a standard practice within the pharmaceutical industry. 

Pharmacologically active allenic steroids have already been examined intensively for about 30 years [5], Thus, the only naturally occurring allenic steroid 107 had been synthesized 3 years before its isolation from Callyspongia diffusa and it had been identified as an inhibitor of the sterol biosynthesis of the silkworm Bombyx mori (Scheme 18.34) [86d], At this early stage, allenic 3-oxo-5,10-secosteroids of type 108 were also used for the irreversible inhibition of ketosteroid isomerases in bacteria, assuming that their activity is probably caused by Michael addition of a nucleophilic amino acid side chain of the enzyme at the 5-position of the steroid [103, 104]. Since this activity is also observed in the corresponding /3,y-acetylenic ketones, it can be rationalized that the latter are converted in vivo into the allenic steroids 108 by enzymatic isomerization [104, 105],... 

Allenic amino acids belong to the classical suicide substrates for the irreversible mechanism-based inhibition of enzymes [5], Among the different types of allenic substrates used for enzyme inhibition [128, 129], the deactivation of vitamin B6 (pyr-idoxal phosphate)-dependent decarboxylases by a-allenic a-amino acids plays an important role (Scheme 18.45). In analogy with the corresponding activity of other /3,y-unsaturated amino acids [102,130], it is assumed that the allenic amino acid 139 reacts with the decarboxylase 138 to furnish the imine 140, which is transformed into a Michael acceptor of type 141 by decarboxylation or deprotonation. Subsequent attack of a suitable nucleophilic group of the active site then leads to inhibition of the decarboxylase by irreversible formation of the adduct 142 [131,132]. 

Free tryptophan is transported into the brain and nerve terminal by an active transport system which it shares with tyrosine and a number of other essential amino acids. On entering the nerve terminal, tryptophan is hydroxylated by tryptophan hydroxylase, which is the rate-limiting step in the synthesis of 5-HT. Tryptophan hydroxylase is not bound in the nerve terminal and optimal activity of the enzyme is only achieved in the presence of molecular oxygen and a pteridine cofactor. Unlike tyrosine hydroxylase, tryptophan hydroxylase is not usually saturated by its substrate. This implies that if the brain concentration rises then the rate of 5-HT synthesis will also increase. Conversely, the rate of 5-HT synthesis will decrease following the administration of experimental drugs such as para-chlorophenylalanine, a synthetic amino acid which irreversibly inhibits the enzyme. Para-chloramphetamine also inhibits the activity of this enzyme, but this experimental drug also increases 5-HT release and delays its reuptake thereby leading to the appearance of the so-called "serotonin syndrome", which in animals is associated with abnormal movements, body posture and temperature. 

Irreversible inhibition of the enzyme leads to accumulation of acetylcholine at these synapses and conseguent neurologic impairment. 

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