Suicide inhibition example

In the preceding section, four diagnostic tests of affinity labeling were listed (inactivation inhibited by substrates, pH dependence of inactivation similar to that of catalysis, labeled inhibitor covalently bound in 1 1 stoichiometry, and saturation kinetics obeyed). The same criteria may be used to diagnose suicide inhibition. In addition, tests must be made to detect any diffusion of the activated intermediate I into solution. For example, the addition of —SH reagents that rapidly react with electrophiles and hence scavenge them should not slow down the rate of reaction. The suicide inhibitor should not, in any case, react with the thiol at an appreciable rate in the absence of enzyme. 

Enzymes containing pyridoxal phosphate are prime targets for suicide inhibition because the chemistry is so naturally suitable. As discussed in Chapter 2, section C2, the pyridoxal ring acts as an electron sink that facilitates the formation of carbanions and also forms part of an extended system of conjugated double bonds. For example, vinyl glycine, CH2=CHCH(NH3+)C02, condenses with the pyridoxal phosphate of aspartate aminotransferase to form a Schiff base, as described in Chapter 2, equation 2.42.19 The a proton may be abstracted (as in equation 2.43) so that the isomerization shown in equation 9.13 readily occurs. 

A classic example of suicide inhibition is that of /3-hydroxy l-decanoyl-dehydrase by 3 -decenoy 1-N-acetylcy steamine.12 The enzyme catalyzes the reaction... 

Mechanism-based inactivation of CYP450 (or suicide inhibition) occurs when a non-toxic drug is metabolised by CYP450 to generate a metabolite that can bind irreversibly with the enzyme. The mechanism of inhibition usually involves free-radical alkylation or acylation of the active site and results in destruction of enzyme activity. Examples of drugs that act in this way include the antibiotic chloramphenicol and the anticancer agent cyclophosphamide. 

Administration of allopurinol, an analog of hypoxanthine, is one treatment for gout. The mechanism of action of allopurinol is interesting it acts first as a substrate and then as an inhibitor of xanthine oxidase. The oxidase hydroxylates allopurinol to alloxanthine (oxipurinol), which then remains tightly bound to the active site. The binding of alloxanthine keeps the molybdenum atom of xanthine oxidase in the + 4 oxidation state instead of it returning to the + 6 oxidation state as in a normal catalytic cycle. We see here another example of suicide inhibition. 

Scheme 4.33 Mechanism of the suicide inhibition of tymidylate synthase by 5-fluorouracil. The reaction pathway with the natural substrate (dUMP) is depicted on the left, the analogous sequence with 5-fluoro-dUMP on the right. The key to the irreversible blocking of the enzyme reaction site is the inability of fluorine to functionally replace hydrogen in proton-transfer reactions, for example the -elimination liberating the enzyme thiolate group [10], In addition, the transient positive charge on the methylene group during hydride transfer is destabilized by the jff-fluorine.

Suicide inhibition is a form of irreversible enzyme inhibition that occurs when an enzyme binds a substrate analog and forms an irreversible complex with it through a covalent bond during the normal catalysis reaction. One example is penicillin, which inhibits transpeptidase from building bacterial cell wall. The mechanism can be given as follows ... 

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. 

Alcohol has a triphasic effect on the elimination rates of drugs that require extensive biotransformation (21). For example, acute alcohol ingestion in combination with a TCA in a teetotaler who attempts suicide will significantly block the first-pass metabolism of the TCA. This chemical inhibition can triple the peak concentration of a TCA by increasing its bioavailability. This is why the consumption of alcohol in association with a TCA overdose increases lethality. 

Reactive metabolites of xenobiotics may differ in reactivity, and therefore have varying impact on enzymatic activities in terms of proximity to their origin. For example, some intermediates are highly reactive and directly inhibit the enzyme that leads to their formation. These substances are commonly referred to as suicide inhibitors, for obvious reasons. Some suicide inhibitors, such as piperonyl butoxide (PBO), a pesticide synergist) are common inhibitors of certain CYP isozymes. PBO amplifies the toxicity of certain insecticides by inhibiting the insect s CYP enzymes that are involved in its degradation. It is metabolized to a highly reactive carbene, which forms an inhibitor complex with the heme iron of CYP, as shown in Scheme 3.6. 

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... 

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). 

Several plant proteins have been isolated that inhibit the metalloprotease carboxypeptidase A [205-217] (Table 7), notably potato carboxypeptidase inhibitor PCI [207-217] (Table 7). PCI is a small, cysteine-rich protein with a compact knotted structure determined by 3 disulphide links. The C-terminal region inserts into the active site of the carboxypeptidase. The C-terminal glycine is cleaved and remains trapped in the active site, this representing an example of suicide inactivation [207-216]. 

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... 

A final group of covalent small-molecule inhibitors of proteases are mechanism-based inhibitors. These inhibitors are enzyme-activated irreversible inhibitors, and they involve a two-hif mechanism that completely inhibits the protease. Some isocoumarins and -lactam derivatives have been shown to be mechanistic inhibitors of serine proteases. A classic example is the inhibition of elastase by several cephalosporin derivatives developed at Merck (Fig. 8). The catalytic serine attacks and opens the -lactam ring of the cephalosporin, which through various isomerization steps, allows for a Michael addition to the active site histidine and the formation of a stable enzyme-inhibitor complex (34). These mechanism-based inhibitors require an initial acylation event to take place before the irreversible inhibitory event. In this way, these small molecules have an analogous mechanism of inhibition to the naturally occurring serpins and a-2-macroglobin, which also act as suicide substrates. 

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