## 4.2.1. Protein Inactivation

Author: Timo Hamers

Reviewers: Frank van Belleghem and Ludek Blaha

Learning objectives:

You should be able to

- discuss how a compound that binds to a protein may inhibit ligand binding, and thereby hamper the function of the protein
- explain the mechanism of action of organophosphate insecticides inhibiting acetylcholinesterase
- explain the mechanism of action of halogenated phenols inhibiting thyroid hormone transport by transthyretin
- distinguish between reversible and irreversible protein inactivation
- distinguish between competitive, non-competitive, and uncompetitive enzyme inhibition

Key words: enzyme inhibition; acetylcholinesterase, transthyretin, competitive inhibition, non-competitive inhibition, uncompetitive inhibition

Introduction

Proteins play an important role in essential biochemical processes including catalysis of metabolic reactions, DNA replication and repair, transport of messengers (e.g. hormones), or receptor responses to such messengers. Many toxic compounds exert their toxic action by binding to a protein and thereby disturbing these vital protein functions.

Inhibition of the protein transport function

Binding of xenobiotic compounds to a transporter protein may hamper binding of the natural ligand of the protein, thereby inhibiting the transporter function of the protein. An example of such inhibition is the binding of halogenated phenols to transthyretin (TTR). TTR is a transport protein for thyroid hormones, present in the blood. It has two binding places for the transport of thyroid hormone, i.e. mainly thyroxine (T4) in mammals and mainly triiodothyronine (T3) in other vertebrates (Figure 1). Compounds with high structural resemblance with thyroid hormone (especially halogenated phenols, such as hydroxylated metabolites of PCBs or PBDEs), are capable to compete with thyroid hormone for TTR binding. Apart from the fact that this enhances distribution of the toxic compounds, this also causes an increase of unbound thyroid hormone in the blood, which is then freely available for uptake in the liver, metabolic conjugation, and urinary excretion. Ultimately, this may lead to decreased thyroid hormone levels in the blood.

Figure 1: Structural resemblance between T4, a hydroxylated PCB metabolite (4-OH-CB-107) and a hydroxylated PBDE metabolite (3-OH-BDE-47). The lower panel illustrates how halogenated phenols (red; e.g. OH-PCB), given their structural resemblance with T4, can compete with T4 (cyan) for TTR-binding (pink), thereby increasing the levels of unbound T4.

Inhibition of the protein enzymatic activity

Proteins involved in the catalysis of a metabolic reaction are called enzymes. The general formula of such a reaction is

Binding of a toxic compound to an enzyme usually causes an inhibition of the enzyme activity, i.e. a decrease in the conversion rate of the endogenous substrate(s) of the enzyme into its/their corresponding product(s). In practice, this causes a toxic response due to a surplus of substrate and/or a deficit of product. One of the classical examples of enzyme inhibition by toxic compounds is the inhibition of the enzyme acetylcholinesterase (AChE) by organophosphate insecticides. AChE catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh), in the cholinergic synapses. During transfer of an action potential from one cell to the other, ACh is released in these synapses from the presynaptic cell into the synaptic cleft in order to stimulate the acetylcholine-receptor (AChR) on the membrane of the postsynaptic cell. AChE, which is also present in these synapses, is then responsible to break down the ACh into acetic acid and choline:

By covalent binding to serine residues in the active site of the AChE enzyme, organophosphate insecticides can inhibit this reaction causing accumulation of the ACh neurotransmitter in the synapse (Fig. 2). As a consequence, the AChR is overstimulated causing convulsions, hypertension, muscle weakness, salivation, lacrimation, gastrointestinal problems, and slow heartbeat.

Figure 2: ACh (blue) is released from the presynaptic neuron into the synapse where it merges to and activates the AChR present on membrane of the postsynaptic cell (not shown). Meanwhile, AChE (grey) present in the synaptic cleft hydrolyses the ACh neurotransmitter to avoid overstimulation of the postsynaptic membrane. Organophosphate insecticides (red) bind to the AChE and prevent its reaction with ACh, causing accumulation of ACh.

Irreversible vs reversible enzyme inhibition

Organophosphate insecticides bind covalently to the AChE enzyme thereby causing irreversible enzyme inhibition. Irreversible enzyme inhibition progressively increases in time following first-order kinetics (link to section on Bioaccumulation and kinetic modelling). Recovery of enzyme activity can only be obtained by de novo synthesis of enzymes. In contrast to AChE inhibition, inhibition of the T4 transport function of TTR is reversible because the halogenated phenols bind to TTR in a non-covalent way. Similarly, non-covalent binding of a toxic compound to an enzyme causes reversible inhibition of the enzyme activity.

In addition to covalent and non-covalent enzyme binding, irreversible enzyme inhibition may occur when toxic compounds cause an error during enzyme synthesis. For instance, ions of essential metals, which are present as cofactors in the active site of many enzymes, may be replaced by ions of other metals during enzyme synthesis, yielding inactive enzymes. A classic example of such decreased enzyme activity is the inhibition of δ-aminolevulinic acid dehydratase (δ-ALAD) by lead. In this case, lead replaces zinc in the active site of the enzyme, thereby inhibiting a catalytic step in the synthesis of a precursor of heme, a cofactor of the protein hemoglobulin (link to section on Toxicity mechanisms of metals).

With respect to reversible enzyme inhibition, three types of inhibition can be distinguished, i.e. competitive, non-competitive, and uncompetitive inhibition (Figure 3).

Figure 3: Three types of reversible enzyme inhibition, i.e. competitive (left), non-competitive (middle), and uncompetitive (right) binding. See text for further explanation. Source: juang.bst.ntu.edu.tw/files/Enz04%20inhibition.PPT

Competitive inhibition refers to a situation where the chemical competes (“fights”) with the substrate for binding to the active site of the enzyme. Competitive inhibition is very specific, because it requires that the inhibitor resembles the substrate and fits in the same binding pocket of the active site. The TTR-binding example described above is a typical example of competitive inhibition between thyroid hormone and halogenated phenols for occupation of the TTR-binding site. A more classic example of competitive inhibition is the inhibition of beta-lactamase by penicillin. Beta-lactamase is an enzyme responsible for the hydrolysis of beta-lactam, which is the final step in bacterial cell wall synthesis. By defective cell wall synthesis, penicillin is an antibiotic causing bacterial death.

Non-competitive inhibition refers to a situation where the chemical binds to an allosteric site of the enzyme (i.e. not the active site), thereby causing a conformational change of the active site. As a consequence, the substrate cannot enter the active site, or the active site becomes inactive, or the product cannot be released from the active site. For instance, echinocandin antifungal drugs non-competitively inhibit the enzyme 1,3-beta glucan synthase, which is responsible for the synthesis of beta-glucan, a major constituent of the fungal cell wall. Lack of beta-glucan in fungal cell walls prevents fungal resistance against osmotic forces, leading to cell lysis.

Uncompetitive inhibition refers to a situation where the chemical can only bind to the enzyme if the substrate is simultaneously bound. Substrate binding leads to a conformational change of the enzyme, which leads to the formation of an allosteric binding site for the inhibitor. Uncompetitive inhibition is more common in two-substrate enzyme reactions than in one-substrate enzyme reactions. An example of uncompetitive inhibition is the inhibition by lithium of the enzyme inositol mono phosphatase (IMPase), which is involved in recycling of the second messenger inositol-3-phospate (I3P) (link to section on Receptor interaction). IMPase is involved in the final step of dephosphorylating inositol monophosphate into inositol. Since lithium is the primary treatment for bipolar disorder, this observation has led to the inositol depletion hypothesis that inhibition of inositol phosphate metabolism offers a plausible explanation for the therapeutic effects of lithium.