Suicide enzyme inhibition, type

The enzymes used to generate reactive quinone methides often undergo inactivation by addition of this electrophile to essential nucleophilic amino acid side chains of the protein catalyst. This is a type of suicide enzyme inhibition.80 This was observed for the acid phosphatase and ribonuclease catalysts used to generate 43.76 79 Alkaline phosphatase has been used to remove the phosphate protecting group from a derivative of an o-difluoromethyl phenyl phosphate that was covalently attached to a solid support. Breakdown of the immobilized 4-hydroxybenzyl difluoride gives an immobilized quinone methide that, in principle, will react irreversibly with proteins and lead to their attachment to the solid support.81... 

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]. 

A second type of CYP enzyme inhibition is mechanism-based inactivation (or suicide inactivation). In this type of inhibition, the effector compound (i.e., the in-... 

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. 

SCHEME 11.3 Postulated mechanisms for the inhibition of serine proteases by coumarin derivatives. NuH nucleophile. Pathway a suicide-type inactivation (suicide substrate). Pathway b transient inactivation by formation of a stable acyl-enzyme (alternate substrate-inhibitor). 

However, when an irreversible inhibitor binds to the enzyme, it cannot be displaced by the substrate and thus binds irreversibly (Fig. 2—51). The irreversible type of enzyme inhibitor is sometimes called a suicide inhibitor because it covalently and irreversibly binds to the enzyme protein, permanently inhibiting it and therefore essentially killing the enzyme by making it nonfunctional forever (Fig. 2—51). Enzyme activity in this case is only restored when new enzyme molecules are synthesized. 

Inhibition can be reversible when it simply complexes at the active site preventing further catalysis. The active enzyme under these conditions can be recovered by dialysis. Another form of inhibition is the irreversible type where the active enzyme cannot be recovered by dialysis. A variant of this type of inhibition is suicide inhibition a substrate of the enzyme reacts at the active site to yield an irreversible inhibitor which then reacts directly with groups at the active site [18]. A technique, in situ click chemistry , is related to that of suicide inhibition and involves click chemistry components which complex at the active site of an enzyme and combine to form femtomolar inhibitors. The technique can be used to synthesise inhibitors or by selection from a library of click chemistry components to search structure space of the inhibitor for the drug target [ 19]. 

Mechanism-based inhibitors or suicide substrates seem to be particularly prevalent with CYP3A4. Such compounds are substrates for the enzyme, but metabolism is believed to form products that deactivate the enzyme. Several macrolide antibiotics, generally involving a tertiary amine function, are able to inhibit CYP3A4 in this manner (147,148). Erythromycin is one of the most widely used examples of this type of interaction, although there are other commonly prescribed agents that inactivate CYP3A4 (149-151), and a consideration of this phenomenon partially explains a number of interactions that are not readily explained by the conventional in vitro data (152). 

The antibiotic chloramphenicol is oxidized by CYP monooxygenase to chloramphenicol oxamyl chloride formed by the oxidation of the dichloromethyl moiety of chloramphenicol followed by elimination of hydrochloric acid " (Figure 33.6). The reactive metabolite reacts with the e-amino group of a lysine residue in CYP and inhibits the enzymatic reaction progressively with time. This type of inhibition is a time-dependent inhibition or a mechanism-based inhibition or inactivation, and the substrate involved historically has been called a suicide substrate because the enzymatic reaction yields a reactive metabolite, which destroys the enzyme. ... 

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. 

The major advantage of suicide inhibitors over competitive inhibitors or other types of irreversible inhibitors is their specificity, because mechanism-based inhibitors usually inhibit only the one enzyme or group of enzymes which had activated them. ... 

Interleukin-lp converting enzyme (ICE, which converts the M, 33,000 protease form of interleukin-ip to the active M, 17,500 form by cleaving after Asp residues) and other members of the ICE-like family of proteases appear to play a significant role in A. For example, expresrion of CrmA protein (a protease inhibitor that inhibits ICE) potently blocks the A. induced by stimulation of Fas or TNF-1, implying that ICE-type proteases are involved in these suicide pathways. Proteins identified as early targets or death substrates associated with the onset of A. are poly(ADP-ribose) polymerase, lamin Bl, a-fodrin, topoisomerase I, p-actin, and the Af, 70,000 compo nent of the U1 small ribonucleoprotein (Ul-70kD). 

Tissues and cultured cells accumulate predominantly shikimate, rather than S-3-P, in the presence of glyphosate, and in cultured buckwheat cells shikimate was shown to accumulate in the vacuole.As neither S-3-P nor shikimate compete with glyphosate for binding sites on EPSP synthase, and as PEP does not accumulate in glyphosate-treated cells (Amrhein, unpublished), the inhibition of EPSP synthase is not overcome. This example illustrates that in order to effectively inhibit an enzyme vivo the inhibitor need not necessarily be of the tight-binding or suicide (Kcat active-site-directed irreversible) type. 

One way to increase selectivity and duration of action of enzyme inhibitors is to design a substrate with a latent electrophile, which becomes unmasked only after it reacts in the active site. This type of inhibition is referred to as suicide or mechanism-based inhibition (1-5) and is, in principle, extremely selective. 

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