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Enzyme Inhibition

Enzyme inhibitors are molecular agents that interfere with catalysis, slowing or halting enzymatic reactions. Enzymes catalyze virtually all cellular processes, so it should not be surprising that enzyme inhibitors are among the most important pharmaceutical agents known. For example, aspirin (acetylsalicylate) inhibits the enzyme that catalyzes the first step in the synthesis of prostaglandins, compounds involved in many processes, including some that produce pain. The study of enzyme inhibitors also has provided valuable information about enzyme mechanisms and has helped define some metabolic pathways. There are two broad classes of enzyme inhibitors: reversible and irreversible.

6.1

Reversible Inhibition

Reversible inhibitors bind to enzymes with non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to produce strong and specific binding. In contrast to substrates and irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can easily be removed by dilution or dialysis. There are four kinds of reversible enzyme inhibition, namely (1) Competitive

inhibition, (2) Uncompetitive inhibition, (3) Mixed inhibition, and Noncompetitive inhibition. They are classified according to the effect of varying the concentration of the enzyme's substrate on the inhibitor.

6.1.1

Competitive Inhibition

6.1.1.1 Mechanism of Competitive Inhibition One common type of reversible inhibition is called competitive inhibition (Figure 6.1a). A competitive inhibitor competes with the substrate for the active site of an enzyme. While the inhibitor (I) occupies the active site it prevents binding of the substrate to the enzyme. This type of inhibition can be overcome by sufficiently high concentrations of 1

substrate (Vmax remains constant), i.e., by out-competing the inhibitor (Figure 6.2). However, the apparent Km (Km app) will increase as it takes a higher concentration of the substrate to reach the Km point, or half the Vmax. Many competitive inhibitors are compounds that resemble the substrate and combine with the enzyme to form an EI complex, but without leading to catalysis.

Figure 6.1: Reversible inhibition. (a) Competitive inhibitors bind to the enzyme’s active site. (b) Uncompetitive inhibitors bind at a separate site, but bind only to the ES complex. Ki is the equilibrium constant for inhibitor binding to E; Ki’ is the equilibrium constant for inhibitor binding to ES. (c) Mixed inhibitors bind at a separate site, but may bind to either E or ES. (d) Noncompetitive inhibitors bind at a separate site, but may bind to either E or ES with an identical affinity.

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Figure 6.2: Competitive inhibition. Because the inhibitor binds reversibly to the enzyme, the competition can be biased to favor the substrate simply by adding more substrate. When [S] far exceeds [I], the probability that an inhibitor molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax. However, the [S] at which vi = ½ Vmax, the apparent Km, increases in the presence of inhibitor by the factor  . This effect on apparent Km, combined with the absence of an effect on Vmax, is diagnostic of competitive inhibition. (The apparent Km app increases in the presence of inhibitor (Km app  Km), while the Vmax remains unchanged.)

6.1.1.2 Substrate Analogs as the Competitive Inhibiters The effects of competitive inhibitors can be overcome by raising the concentration of the substrate. Most frequently, in competitive inhibition the inhibitor, I, binds to the substrate-binding portion of the active site and blocks access by the substrate. The structures of most classic competitive inhibitors therefore tend to resemble the structures of a substrate and thus are termed substrate analogs. Inhibition of the enzyme succinate dehydrogenase by malonate illustrates competitive inhibition by a substrate analog. Succinate dehydrogenase catalyzes the removal of one hydrogen atom from each of the two methylene carbons of succinate (Figure 6.3). Both succinate and its structural analog malonate (OOCCH2COO) can bind to the active site of succinate dehydrogenase, forming an ES or an EI complex, respectively. However, since malonate contains only one methylene carbon, it cannot undergo dehydrogenation.

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Figure 6.3: The succinate dehydrogenase reaction.

6.1.1.3 Medical Therapy Based on Competitive Inhibition Treatment of Bacterial Infections: Sulfonamides or Sulfa Drugs Competitive inhibitors are important in the treatment of many microbial diseases. Sulfa drugs like sulfanilamide resemble p-aminobenzoate, a molecule used in the formation of the coenzyme folic acid. These analogues compete with metabolites in metabolic processes because of their similarity, but are just different enough so that they cannot function normally in cellular metabolism. The first antimetabolites to be used successfully as chemotherapeutic agents were the sulfonamides, discovered by G Domagk. Sulfonamides or sulfa drugs are structurally related to sulfanilamide, an analogue of p-aminobenzoic acid (Figure 6.4). The latter substance is used in the synthesis of the cofactor folic acid. When sulfanilamide or another sulfonamide enters a bacterial cell, it competes with p-aminobenzoic acid for the active site of an enzyme involved in folic acid synthesis, and the folate concentration decreases.

Figure 6.4: Sulfanilamide and its relationship to the structure of folic acid. 4

The decline in folic acid is detrimental to the bacterium because folic acid is essential to the synthesis of purines and pyrimidines, the bases used in the construction of DNA, RNA, and other important cell constituents. The resulting inhibition of purine and pyrimidine synthesis leads to cessation of bacterial growth or death of the pathogen. Sulfonamides are selectively toxic for many pathogens because these bacteria manufacture their own folate and cannot effectively take up the cofactor. In contrast, humans cannot synthesize folate and must obtain it in the diet; therefore sulfonamides will not affect the host. Treatment of Methanol Intoxication A medical therapy based on competition at the active site is used to treat patients who have ingested methanol, a solvent found in gas-line antifreeze. The liver enzyme alcohol dehydrogenase converts methanol to formaldehyde, which is damaging to many tissues. Blindness is a common result of methanol ingestion, because the eyes are particularly sensitive to formaldehyde. Ethanol competes effectively with methanol as an alternative substrate for alcohol dehydrogenase. The effect of ethanol is much like that of a competitive inhibitor, with the distinction that ethanol is also a substrate for alcohol dehydrogenase and its concentration will decrease over time as the enzyme converts it to acetaldehyde. The therapy for methanol poisoning is slow intravenous infusion of ethanol, at a rate that maintains a controlled concentration in the bloodstream for several hours. This slows the formation of formaldehyde, lessening the danger while the kidneys filter out the methanol to be excreted harmlessly in the urine.

6.1.1.4 Quantitatively Analysis of Competitive Inhibition Competitive inhibition can be analyzed quantitatively by steady-state kinetics. In the presence of a competitive inhibitor, the Michaelis-Menten equation (Equation 1) becomes (Equation 2):

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where

The Equation 2 describes the important features of competitive inhibition. The experimentally determined variable  Km, the Km observed in the presence of the inhibitor, is often called the “apparent” Km (Km app) (Figure 6.2). The velocity equation for competitive inhibition in reciprocal form is (Equation 3):

Equation 2 and 3 describes the important features of competitive inhibition. The experimentally determined variable Km, the Km observed in the presence of the inhibitor, is often called the “apparent” Km. Because the inhibitor binds reversibly to the enzyme, the competition can be biased to favor the substrate simply by adding more substrate. When [S] far exceeds [I], the probability that an inhibitor molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax (Figure 6.2). However, the [S] at which v0 = ½ Vmax, the apparent Km, increases in the presence of inhibitor. This effect on apparent Km, combined with the absence of an effect on Vmax, is diagnostic of competitive inhibition and is readily revealed in a double reciprocal plot (Figure 6.5). The equilibrium constant for inhibitor binding (Ki) can be obtained from the same plot.

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Figure 6.5: Lineweaver-Burk plot of competitive inhibition. Note the complete relief of inhibition at high [S] (i.e., low 1/[S]).

6.1.2 Uncompetitive Inhibition 6.1.2.1 Mechanism of Uncompetitive Inhibition An uncompetitive inhibitor binds at a site distinct from the substrate active site and, unlike a competitive inhibitor, binds only to the ES complex (Figure 6.1-b). This type of inhibition causes Vmax to decrease (maximum velocity decreases as a result of removing activated complex) and Km to decrease (due to better binding efficiency and the effective elimination of the ES complex thus decreasing the Km, which indicates a higher binding affinity).

6.1.2.2 Quantitatively Analysis of Uncompetitive Inhibition In the presence of an uncompetitive inhibitor, the Michaelis-Menten equation is altered to (Equation 4):

where

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As described by the Equation 3, at high concentrations of substrate, vi approaches Vmax/ . Thus, an uncompetitive inhibitor lowers the measured Vmax (Figure 6.6). Apparent Km also decreases, because the [S] required to reach one-half Vmax decreases by the factor .

Figure 6.6: Uncompetitive inhibition. Initial velocity (vi) versus substrate concentration [S] plot in the presence of an uncompetitive inhibitor. Both Vmax and Km decrease by an uncompetitive inhibitor to the same extent (Vmax app  Vmax; Km app  Km). The reciprocal form of the velocity equation for uncompetitive inhibition is (Equation 5):

Both Vmax and Km decrease by an uncompetitive inhibitor to the same extent. The vi versus [S] plot and 1/vi versus 1/[S] is shown in Figure 6.7.

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Figure 6.7: (a) v versus [S] plot in the presence of an uncompetitive inhibitor. (b) 1/vi versus 1/[S] plot in the presence of fixed concentration of an uncompetitive inhibitor. Both Vmax and Km decrease by an uncompetitive inhibitor to the same extent. ’

6.1.3 Mixed Inhibition 6.1.3.1 Mechanism of Mixed Inhibition A mixed inhibitor can bind to the enzyme at the same time as the enzyme's substrate. However, the binding of the inhibitor affects the binding of the substrate, and vice versa. This type of inhibition can be reduced, but not overcome by increasing concentrations of substrate. Mixed inhibition refers to a combination of two different types of reversible enzyme inhibition — competitive inhibition and uncompetitive inhibition. The term 'mixed' is used when the inhibitor can bind to either the free enzyme or the enzymesubstrate complex. In mixed inhibition, the inhibitor binds to a site different from the active site where the substrate binds. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e., tertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced. However, not all inhibitors that bind at allosteric sites are mixed inhibitors. Mixed inhibition may result in either a decrease in the apparent affinity of the enzyme for the substrate (Km app  Km, a decrease in apparent affinity means the Km value appears to increase) in cases where the inhibitor favors binding the free enzyme (Figure 6.8), or 9

in an increase in the apparent affinity (Km

app

 Km), an increase in apparent affinity

means the Km value appears to decrease) when the inhibitor binds favorably to the enzyme-substrate complex. In either case the inhibition decreases the apparent maximum enzyme reaction rate (Vmax app  Vmax).

Figure 6.8: Mixed inhibition. Initial velocity (vi) versus substrate concentration [S] plot in the presence of an mixe inhibitor. Mixed-type inhibitors bind to both E and ES, but their affinities for these two forms of the enzyme are different (Ki ≠ Ki'). Thus, mixed-type inhibitors interfere with substrate binding (increase Km) and hamper catalysis in the ES complex (decrease Vmax).

6.1.3.2 Quantitatively Analysis of Mixed Inhibition The rate equation (the Michaelis-Menten equation) describing mixed inhibition is (Equation 6):

where  and  are defined as above. A mixed inhibitor usually affects both Km and Vmax. The reciprocal form (Figure 6.9) of the velocity equation for uncompetitive inhibition is (Equation 7):

A mixed inhibitor usually affects both Km and Vmax. However, the binding of the inhibitor affects the binding of the substrate, and vice versa. This type of inhibition can 10

be reduced, but not overcome by increasing concentrations of substrate. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e., tertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced.

Figure 6.9: 1/v versus 1/[S] plot in the presence of fixed concentration of an uncompetitive inhibitor.

6.1.4 Noncompetitive Inhibition 6.1.4.1 Mechanism of Noncompetitive Inhibition In noncompetitive inhibition, the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. In noncompetitive inhibition, the ability of the inhibitor to bind the enzyme is exactly the same whether or not the enzyme has already bound the substrate. Noncompetitive inhibition is sometimes thought of as a special case of mixed inhibition (where  =  ), which rarely encountered in experiments, and classically has been defined as noncompetitive inhibition. Also in case of the mixed inhibition, the inhibitor binds to an allosteric site, i.e., a site different from the active site where the substrate binds. As a result, the extent of inhibition depends only on the concentration of the inhibitor. Unlike mixed inhibition, noncompetitive inhibition does not change Km (i.e., it does not affect substrate binding) but only

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decreases Vmax (i.e., inhibitor binding hampers catalysis). Vmax will decrease due to the inability for the reaction to proceed as efficiently, but Km will remain the same as the actual binding of the substrate, by definition, will still function properly (Figure 6.10). The most common mechanism of noncompetitive inhibition involves reversible binding of the inhibitor to an allosteric site, but it is possible for the inhibitor to operate via other means including direct binding to the active site. It differs from competitive inhibition in that the binding of the inhibitor does not prevent binding of substrate, and vice versa, it simply prevents product formation. This type of inhibition reduces the maximum rate of a chemical reaction without changing the apparent binding affinity of the catalyst for the substrate. In the presence of a noncompetitive inhibitor, the apparent enzyme affinity is equivalent to the actual affinity. In terms of MichaelisMenten kinetics, Km app = Km (Figure 6.10).

Figure 6.10: Initial velocity (vi) versus substrate concentration [S] plot in the presence of an noncompetitive inhibitor. Noncompetitive inhibitors have identical affinities for E and ES (Ki = Ki'). Noncompetitive inhibition does not change Km (i.e., it does not affect substrate binding) but decreases Vmax (i.e., inhibitor binding hampers catalysis).

6.1.4.2 Quantitatively Analysis of Noncompetitive Inhibition The rate equation describing noncompetitive inhibition is same as the mixed inhibition (Equation 6):

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where  and  are defined as above. While a mixed inhibitor usually affects both Km and Vmax, but a noncompetitive inhibitor affects only the Vmax (decreased in Vmax). The reciprocal form (Figure 6.11) of the velocity equation for uncompetitive inhibition is same as the mixed inhibition (Equation 7):

Figure 6.11: Plots of v versus [S] for noncompetitive inhibition (Lineweaver-Burk plot for simple noncompetitive inhibition).

6.2

Irreversible Inhibition

6.2.1

Mechanism of Irreversible Inhibition

Irreversible inhibitors usually covalently modify an enzyme, and inhibition can therefore not be reversed. Irreversible inhibitors often contain reactive functional groups such as nitrogen mustards, aldehydes, haloalkanes, alkenes, Michael acceptors, phenyl sulfonates, or fluorophosphonates. These electrophilic groups react with amino acid side chains to form covalent adducts. The residues modified are those with side chains containingnucleophiles such as hydroxyl or sulfhydryl groups; these include the amino acids serine (as in di-isopropylphosphofluoridate or DFP), cysteine, threonine, or tyrosine.

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Irreversible inhibition is different from irreversible enzyme inactivation. Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying protein structure but by specifically altering the active site of their target. For example, extremes of pH or temperature usually cause denaturation of all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical treatments destroy protein structure: for example, heating in concentrated hydrochloric acid will hydrolyze the peptide bonds holding proteins together, releasing free amino acids.

6.2.2 Examples of Irreversible Inhibitors Enzyme inhibitors are often poisonous. For example, di-isopropylfluorophosphate is a nerve poison because the enzyme acetylcholinesterase in the synapses of neuron has a reactive site serine (Figure 6.12), and consequently is a potent neurotoxin, with a lethal dose of less than 100 mg. Chymotrypsin and acetylcholinesterase are both members of the class of enzymes known as serine esterases, which are all inhibited by di-isopropylfluorophosphate.

Figure 6.12: Reaction of the irreversible inhibitor in di-isopropylphosphofluoridate (DFP) with a serine protease. Di-isopropylfluorophosphate transfers its phosphate to the active site serine. The resulting phospho‐enzyme is totally inactive.

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6.2.3 Suicide Inhibitors (Mechanism-Based Inactivators) Suicide inhibition is an unusual type of irreversible inhibition where the enzyme converts the inhibitor into a reactive form in its active site. An example is the inhibitor of polyamine biosynthesis, -difluoromethylornithine or DFMO, which is an analogue of the amino acid ornithine (Figure 6.13), and is used to treat African trypanosomiasis (sleeping sickness). DFMO is a suicide inhibitor of ornithine decarbooxylase, which, in most organisms, is the rate-limiting enzyme for the synthesis of polyamines from ornithine. In humans, loss of ornithine decarbooxylase function causes an accumulation of ornithine that results in gyrate atrophy of the choroid and retina, a disease that progressively leads to blindness. Ornithine decarboxylase can catalyse the decarboxylation of...