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5A: Introduction to Reversible Binding - Biology

5A: Introduction to Reversible Binding - Biology


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Learning Objectives

  • write equations for the dissociation constant (KD), mass balance of total macromolecule (M0), total ligand (L0), and [ML] as a function of L or Lo ([ML] = [M0][L]/(KD+ [L]) (when Lo >> Mo or when free [L] is known) and Y = fractional saturation = Y = ([ML]/[M0] = [L]/(KD+ [L])
  • decide which of two given equations for [ML] should be used under conditions when the above conditions for L0 and L are given
  • based on the equation ([ML] = [M0][L]/(KD+ [L]) draw qualitative graphs for different given L0, L, and Kd values
  • determine fraction saturation given relatives values of Kd and L, assuming L0 >> M0
  • compare relative % bound for covalent binding of protons to an acid and noncovalent binding of a ligand to a macromolecule given pka/pH and Kd/L values
  • describe differences in binding curves for binding of a ligand to a macromolecule and the dimerization of a macromolecule
  • derive an equation which shows the relationships between the rate constant for binding (kon), dissociation (koff) and the thermodynamic dissociation (Kd) or equilibrium constant (Keq).
  • describe the structural and mathematic differences between specific and nonspecific binding
  • given a Kd, estimate t1/2 values for the lifetime of the ML complex.
  • describe techniques used to determine ML for given L or L0 values, including those that do and do not require separation of ML from M , so that Kd values for a M and L interaction can be determined
  • List advantages of isothermal titration calorimetry and surface plasmon resonance in determination of binding interaction parameters

Cis-regulatory element

Cis-regulatory elements (CREs) or Cis-regulatory modules (CRMs) are regions of non-coding DNA which regulate the transcription of neighboring genes. CREs are vital components of genetic regulatory networks, which in turn control morphogenesis, the development of anatomy, and other aspects of embryonic development, studied in evolutionary developmental biology.

CREs are found in the vicinity of the genes that they regulate. CREs typically regulate gene transcription by binding to transcription factors. A single transcription factor may bind to many CREs, and hence control the expression of many genes (pleiotropy). The Latin prefix cis means "on this side", i.e. on the same molecule of DNA as the gene(s) to be transcribed.

CRMs are stretches of DNA, usually 100–1000 DNA base pairs in length, [1] where a number of transcription factors can bind and regulate expression of nearby genes and regulate their transcription rates. They are labeled as cis because they are typically located on the same DNA strand as the genes they control as opposed to trans, which refers to effects on genes not located on the same strand or farther away, such as transcription factors. [1] One cis-regulatory element can regulate several genes, [2] and conversely, one gene can have several cis-regulatory modules. [3] Cis-regulatory modules carry out their function by integrating the active transcription factors and the associated co-factors at a specific time and place in the cell where this information is read and an output is given. [4]

CREs are often but not always upstream of the transcription site. CREs contrast with trans-regulatory elements (TREs). TREs code for transcription factors. [ citation needed ]


Contents

Binding of a ligand to a binding site on protein often triggers a change in conformation in the protein and results in altered cellular function. Hence binding site on protein are critical parts of signal transduction pathways. [10] Types of ligands include neurotransmitters, toxins, neuropeptides, and steroid hormones. [11] Binding sites incur functional changes in a number of contexts, including enzyme catalysis, molecular pathway signaling, homeostatic regulation, and physiological function. Electric charge, steric shape and geometry of the site selectively allow for highly specific ligands to bind, activating a particular cascade of cellular interactions the protein is responsible for. [12] [13]

Catalysis Edit

Enzymes incur catalysis by binding more strongly to transition states than substrates and products. At the catalytic binding site, several different interactions may act upon the substrate. These range from electric catalysis, acid and base catalysis, covalent catalysis, and metal ion catalysis. [11] These interactions decrease the activation energy of a chemical reaction by providing favorable interactions to stabilize the high energy molecule. Enzyme binding allows for closer proximity and exclusion of substances irrelevant to the reaction. Side reactions are also discouraged by this specific binding. [14] [11]

Types of enzymes that can perform these actions include oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. [15]

For instance, the transferase hexokinase catalyzes the phosphorylation of glucose to make glucose-6-phosphate. Active site residues of hexokinase allow for stabilization of the glucose molecule in the active site and spur the onset of an alternative pathway of favorable interactions, decreasing the activation energy. [16]

Inhibition Edit

Protein inhibition by inhibitor binding may induce obstruction in pathway regulation, homeostatic regulation and physiological function.

Competitive inhibitors compete with substrate to bind to free enzymes at active sites and thus impede the production of the enzyme-substrate complex upon binding. For example, carbon monoxide poisoning is caused by the competitive binding of carbon monoxide as opposed to oxygen in hemoglobin.

Uncompetitive inhibitors, alternatively, bind concurrently with substrate at active sites. Upon binding to an enzyme substrate (ES) complex, an enzyme substrate inhibitor (ESI) complex is formed. Similar to competitive inhibitors, the rate at product formation is decreased also. [5]

Lastly, mixed inhibitors are able to bind to both the free enzyme and the enzyme-substrate complex. However, in contrast to competitive and uncompetitive inhibitors, mixed inhibitors bind to the allosteric site. Allosteric binding induces conformational changes that may increase the protein's affinity for substrate. This phenomenon is called positive modulation. Conversely, allosteric binding that decreases the protein's affinity for substrate is negative modulation. [17]

Active site Edit

At the active site, a substrate binds to an enzyme to induce a chemical reaction. [18] [19] Substrates, transition states, and products can bind to the active site, as well as any competitive inhibitors. [18] For example, in the context of protein function, the binding of calcium to troponin in muscle cells can induce a conformational change in troponin. This allows for tropomyosin to expose the actin-myosin binding site to which the myosin head binds to form a cross-bridge and induce a muscle contraction. [20]

In the context of the blood, an example of competitive binding is carbon monoxide which competes with oxygen for the active site on heme. Carbon monoxide's high affinity may outcompete oxygen in the presence of low oxygen concentration. In these circumstances, the binding of carbon monoxide induces a conformation change that discourages heme from binding to oxygen, resulting in carbon monoxide poisoning. [5]

Allosteric site Edit

At the regulatory site, the binding of a ligand may elicit amplified or inhibited protein function. [5] [21] The binding of a ligand to an allosteric site of a multimeric enzyme often induces positive cooperativity, that is the binding of one substrate induces a favorable conformation change and increases the enzyme's likelihood to bind to a second substrate. [22] Regulatory site ligands can involve homotropic and heterotropic ligands, in which single or multiple types of molecule affects enzyme activity respectively. [23]

Enzymes that are highly regulated are often essential in metabolic pathways. For example, phosphofructokinase (PFK), which phosphorylates fructose in glycolysis, is largely regulated by ATP. Its regulation in glycolysis is imperative because it is the committing and rate limiting step of the pathway. PFK also controls the amount of glucose designated to form ATP through the catabolic pathway. Therefore, at sufficient levels of ATP, PFK is allosterically inhibited by ATP. This regulation efficiently conserves glucose reserves, which may be needed for other pathways. Citrate, an intermediate of the citric acid cycle, also works as an allosteric regulator of PFK. [23] [24]

Single- and multi-chain binding sites Edit

Binding sites can be characterized also by their structural features. Single-chain sites (of “monodesmic” ligands, μόνος: single, δεσμός: binding) are formed by a single protein chain, while multi-chain sites (of "polydesmic” ligands, πολοί: many) [25] are frequent in protein complexes, and are formed by ligands that bind more than one protein chain, typically in or near protein interfaces. Recent research shows that binding site structure has profound consequences for the biology of protein complexes (evolution of function, allostery). [26] [27]

Cryptic binding sites Edit

Cryptic binding sites are the binding sites that are transiently formed in an apo form or that are induced by ligand binding. Considering the cryptic binding sites increases the size of the potentially “druggable” human proteome from

78% of disease-associated proteins. [28] The binding sites have been investigated by: support vector machine applied to "CryptoSite" data set, [28] Extension of "CryptoSite" data set, [29] long timescale molecular dynamics simulation with Markov state model and with biophysical experiments, [30] and cryptic-site index that is based on relative accessible surface area. [31]

Binding curves describe the binding behavior of ligand to a protein. Curves can be characterized by their shape, sigmoidal or hyperbolic, which reflect whether or not the protein exhibits cooperative or noncooperative binding behavior respectively. [32] Typically, the x-axis describes the concentration of ligand and the y-axis describes the fractional saturation of ligands bound to all available binding sites. [5] The Michaelis Menten equation is usually used when determining the shape of the curve. The Michaelis Menten equation is derived based on steady-state conditions and accounts for the enzyme reactions taking place in a solution. However, when the reaction takes place while the enzyme is bound to a substrate, the kinetics play out differently. [33]

Modeling with binding curves are useful when evaluating the binding affinities of oxygen to hemoglobin and myoglobin in the blood. Hemoglobin, which has four heme groups, exhibits cooperative binding. This means that the binding of oxygen to a heme group on hemoglobin induces a favorable conformation change that allows for increased binding favorability of oxygen for the next heme groups. In these circumstances, the binding curve of hemoglobin will be sigmoidal due to its increased binding favorability for oxygen. Since myoglobin has only one heme group, it exhibits noncooperative binding which is hyperbolic on a binding curve. [34]

Biochemical differences between different organisms and humans are useful for drug development. For instance, penicillin kills bacterial enzymes by inhibiting DD-transpeptidase, destroying the development of the bacterial cell wall and inducing cell death. Thus, the study of binding sites is relevant to many fields of research, including cancer mechanisms, [35] drug formulation, [36] and physiological regulation. [37] The formulation of an inhibitor to mute a protein's function is a common form of pharmaceutical therapy. [38]

In the scope of cancer, ligands that are edited to have a similar appearance to the natural ligand are used to inhibit tumor growth. For example, Methotrexate, a chemotherapeutic, acts as a competitive inhibitor at the dihydrofolate reductase active site. [39] This interaction inhibits the synthesis of tetrahydrofolate, shutting off production of DNA, RNA and proteins. [39] Inhibition of this function represses neoplastic growth and improves severe psoriasis and adult rheumatoid arthritis. [38]

In cardiovascular illnesses, drugs such as beta blockers are used to treat patients with hypertension. Beta blockers (β-Blockers) are antihypertensive agents that block the binding of the hormones adrenaline and noradrenaline to β1 and β2 receptors in the heart and blood vessels. These receptors normally mediate the sympathetic "fight or flight" response, causing constriction of the blood vessels. [40]

Competitive inhibitors are also largely found commercially. Botulinum toxin, known commercially as Botox, is a neurotoxin causes flaccid paralysis in the muscle due to binding to acetylcholine dependent nerves. This interaction inhibits muscle contractions, giving the appearance of smooth muscle. [41]

A number of computational tools have been developed for the prediction of the location of binding sites on proteins. [21] [42] [43] These can be broadly classified into sequence based or structure based. [43] Sequence based methods rely on the assumption that the sequences of functionally conserved portions of proteins such as binding site are conserved. Structure based methods require the 3D structure of the protein. These methods in turn can be subdivided into template and pocket based methods. [43] Template based methods search for 3D similarities between the target protein and proteins with known binding sites. The pocket based methods search for concave surfaces or buried pockets in the target protein that possess features such as hydrophobicity and hydrogen bonding capacity that would allow them to bind ligands with high affinity. [43] Even though the term pocket is used here, similar methods can be used to predict binding sites used in protein-protein interactions that are usually more planar, not in pockets. [44]


Contents

Types of reversible inhibitors Edit

Reversible inhibitors attach 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 be easily removed by dilution or dialysis.

There are four kinds of reversible enzyme inhibitors. They are classified according to the effect of varying the concentration of the enzyme's substrate on the inhibitor. [3] [4] [1]

  • In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time, as shown in the figure on the right. This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds the substrate and inhibitor compete for access to the enzyme's active site. This type of inhibition can be overcome by sufficiently high concentrations of substrate (Vmax remains constant), i.e., by out-competing the inhibitor. However, the apparent Km will increase as it takes a higher concentration of the substrate to reach the Km point, or half the Vmax. Competitive inhibitors are often similar in structure to the real substrate (see examples below).
  • In uncompetitive inhibition, the inhibitor binds only to the substrate-enzyme complex. 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 as a result of Le Chatelier's principle and the effective elimination of the ES complex thus decreasing the Km which indicates a higher binding affinity).
  • In non-competitive inhibition, the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. As a result, the extent of inhibition depends only on the concentration of the inhibitor. 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.
  • In mixed inhibition, the 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. 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.

These types can also be distinguished by the effect of increasing the substrate concentration [S] on the degree of inhibition caused by a given amount of inhibitor. For competitive inhibition the degree of inhibition is reduced by increasing [S], for noncompetitive inhibition the degree of inhibition is unchanged, and for uncompetitive (also called anticompetitive) inhibition the degree of inhibition increases with [S]. [6]

Quantitative description of reversible inhibition Edit

Reversible inhibition can be described quantitatively in terms of the inhibitor's binding to the enzyme and to the enzyme-substrate complex, and its effects on the kinetic constants of the enzyme. In the classic Michaelis-Menten scheme below, an enzyme (E) binds to its substrate (S) to form the enzyme–substrate complex ES. Upon catalysis, this complex breaks down to release product P and free enzyme. The inhibitor (I) can bind to either E or ES with the dissociation constants Ki or Ki', respectively.

  • Competitive inhibitors can bind to E, but not to ES. Competitive inhibition increases Km (i.e., the inhibitor interferes with substrate binding), but does not affect Vmax (the inhibitor does not hamper catalysis in ES because it cannot bind to ES).
  • Uncompetitive inhibitors bind to ES. Uncompetitive inhibition decreases both Km' and 'Vmax. The inhibitor affects substrate binding by increasing the enzyme's affinity for the substrate (decreasing Km) as well as hampering catalysis (decreases Vmax).
  • Non-competitive inhibitors have identical affinities for E and ES (Ki = Ki'). Non-competitive inhibition does not change Km (i.e., it does not affect substrate binding) but decreases Vmax (i.e., inhibitor binding hampers catalysis).
  • Mixed-type inhibitors bind to both E and ES, but their affinities for these two forms of the enzyme are different (KiKi'). Thus, mixed-type inhibitors affect substrate binding (increase or decrease Km) and hamper catalysis in the ES complex (decrease Vmax).

When an enzyme has multiple substrates, inhibitors can show different types of inhibition depending on which substrate is considered. This results from the active site containing two different binding sites within the active site, one for each substrate. For example, an inhibitor might compete with substrate A for the first binding site, but be a non-competitive inhibitor with respect to substrate B in the second binding site. [7]

Measuring the dissociation constants of a reversible inhibitor Edit

As noted above, an enzyme inhibitor is characterised by its two dissociation constants, Ki and Ki', to the enzyme and to the enzyme-substrate complex, respectively. The enzyme-inhibitor constant Ki can be measured directly by various methods one extremely accurate method is isothermal titration calorimetry, in which the inhibitor is titrated into a solution of enzyme and the heat released or absorbed is measured. [8] However, the other dissociation constant Ki' is difficult to measure directly, since the enzyme-substrate complex is short-lived and undergoing a chemical reaction to form the product. Hence, Ki' is usually measured indirectly, by observing the enzyme activity under various substrate and inhibitor concentrations, and fitting the data [9] to a modified Michaelis–Menten equation

where the modifying factors α and α' are defined by the inhibitor concentration and its two dissociation constants

Thus, in the presence of the inhibitor, the enzyme's effective Km and Vmax become (α/α')Km and (1/α')Vmax, respectively. However, the modified Michaelis-Menten equation assumes that binding of the inhibitor to the enzyme has reached equilibrium, which may be a very slow process for inhibitors with sub-nanomolar dissociation constants. In these cases, it is usually more practical to treat the tight-binding inhibitor as an irreversible inhibitor (see below) however, it can still be possible to estimate Ki' kinetically if Ki is measured independently.

The effects of different types of reversible enzyme inhibitors on enzymatic activity can be visualized using graphical representations of the Michaelis–Menten equation, such as Lineweaver–Burk plots, Eadie-Hofstee plots or Hanes-Woolf plots. For example, in the Lineweaver–Burk plots at the right, the competitive inhibition lines intersect on the y-axis, illustrating that such inhibitors do not affect Vmax. Similarly, the non-competitive inhibition lines intersect on the x-axis, showing these inhibitors do not affect Km. However, it can be difficult to estimate Ki and Ki' accurately from such plots, [10] so it is advisable to estimate these constants using more reliable nonlinear regression methods, as described above.

Reversible inhibitors Edit

Traditionally reversible enzyme inhibitors have been classified as competitive, uncompetitive, or non-competitive, according to their effects on Km and Vmax. These different effects result from the inhibitor binding to the enzyme E, to the enzyme–substrate complex ES, or to both, respectively. The division of these classes arises from a problem in their derivation and results in the need to use two different binding constants for one binding event. The binding of an inhibitor and its effect on the enzymatic activity are two distinctly different things, another problem the traditional equations fail to acknowledge. In noncompetitive inhibition the binding of the inhibitor results in 100% inhibition of the enzyme only, and fails to consider the possibility of anything in between. [11] The common form of the inhibitory term also obscures the relationship between the inhibitor binding to the enzyme and its relationship to any other binding term be it the Michaelis–Menten equation or a dose response curve associated with ligand receptor binding. To demonstrate the relationship the following rearrangement can be made:

This rearrangement demonstrates that similar to the Michaelis–Menten equation, the maximal rate of reaction depends on the proportion of the enzyme population interacting with its substrate.

fraction of the enzyme population bound by substrate

fraction of the enzyme population bound by inhibitor

the effect of the inhibitor is a result of the percent of the enzyme population interacting with inhibitor. The only problem with this equation in its present form is that it assumes absolute inhibition of the enzyme with inhibitor binding, when in fact there can be a wide range of effects anywhere from 100% inhibition of substrate turn over to just >0%. To account for this the equation can be easily modified to allow for different degrees of inhibition by including a delta Vmax term.

This term can then define the residual enzymatic activity present when the inhibitor is interacting with individual enzymes in the population. However the inclusion of this term has the added value of allowing for the possibility of activation if the secondary Vmax term turns out to be higher than the initial term. To account for the possibly of activation as well the notation can then be rewritten replacing the inhibitor "I" with a modifier term denoted here as "X".

While this terminology results in a simplified way of dealing with kinetic effects relating to the maximum velocity of the Michaelis–Menten equation, it highlights potential problems with the term used to describe effects relating to the Km. The Km relating to the affinity of the enzyme for the substrate should in most cases relate to potential changes in the binding site of the enzyme which would directly result from enzyme inhibitor interactions. As such a term similar to the one proposed above to modulate Vmax should be appropriate in most situations: [12]

Special cases Edit

  • The mechanism of partially competitive inhibition is similar to that of non-competitive, except that the EIS complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of the enzyme–substrate (ES) complex. This inhibition typically displays a lower Vmax, but an unaffected Km value. [13]
  • Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme–substrate complex, not to the free enzyme the EIS complex is catalytically inactive. This mode of inhibition is rare and causes a decrease in both Vmax and the Km value. [13]
  • Substrate and product inhibition is where either the substrate or product of an enzyme reaction inhibit the enzyme's activity. This inhibition may follow the competitive, uncompetitive or mixed patterns. In substrate inhibition there is a progressive decrease in activity at high substrate concentrations. This may indicate the existence of two substrate-binding sites in the enzyme. [1] At low substrate, the high-affinity site is occupied and normal kinetics are followed. However, at higher concentrations, the second inhibitory site becomes occupied, inhibiting the enzyme. [14] Product inhibition is often a regulatory feature in metabolism and can be a form of negative feedback.
  • Slow-tight inhibition occurs when the initial enzyme–inhibitor complex EI undergoes isomerisation to a second more tightly held complex, EI*, but the overall inhibition process is reversible. This manifests itself as slowly increasing enzyme inhibition. Under these conditions, traditional Michaelis–Menten kinetics give a false value for Ki, which is time–dependent. [1] The true value of Ki can be obtained through more complex analysis of the on (kon) and off (koff) rate constants for inhibitor association. See irreversible inhibition below for more information.
  • Bi-substrate analog inhibitors are high affinity and selectivity inhibitors that can be prepared for enzymes that catalyze bi-molecular reactions by capturing the binding energy of each substrate into one molecule. [15][16] For example, in the formyl transfer reactions of purine biosynthesis, a potent multi-substrate adduct inhibitor (MAI) to GAR TFase was prepared synthetically by linking analogs of the glycinamide ribonucleotide (GAR) substrate and the N-10-formyl tetrahydrofolate cofactor together to produce thioglycinamide ribonucleotide dideazafolate (TGDDF), [17] or enzymatically from the natural GAR substrate to yield GDDF. [18] Here the subnanomolar dissociation constant (KD) of TGDDF was greater than predicted presumably due to entropic advantages gained and/or positive interactions acquired through the atoms linking the components. MAIs have also been observed to be produced in cells by reactions of pro-drugs such as isoniazid[19] or enzyme inhibitor ligands (e.g., PTC124) [20] with cellular cofactors such as NADH and ATP respectively.

Examples of reversible inhibitors Edit

As enzymes have evolved to bind their substrates tightly, and most reversible inhibitors bind in the active site of enzymes, it is unsurprising that some of these inhibitors are strikingly similar in structure to the substrates of their targets. Inhibitors of DHFR are prominent examples. Other example of these substrate mimics are the protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV. [21] The structure of ritonavir, a protease inhibitor based on a peptide and containing three peptide bonds, is shown on the right. As this drug resembles the protein that is the substrate of the HIV protease, it competes with this substrate in the enzyme's active site.

Enzyme inhibitors are often designed to mimic the transition state or intermediate of an enzyme-catalyzed reaction. This ensures that the inhibitor exploits the transition state stabilising effect of the enzyme, resulting in a better binding affinity (lower Ki) than substrate-based designs. An example of such a transition state inhibitor is the antiviral drug oseltamivir this drug mimics the planar nature of the ring oxonium ion in the reaction of the viral enzyme neuraminidase. [22]

However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV protease inhibitor tipranavir is shown on the left. This molecule is not based on a peptide and has no obvious structural similarity to a protein substrate. These non-peptide inhibitors can be more stable than inhibitors containing peptide bonds, because they will not be substrates for peptidases and are less likely to be degraded. [23]

In drug design it is important to consider the concentrations of substrates to which the target enzymes are exposed. For example, some protein kinase inhibitors have chemical structures that are similar to adenosine triphosphate, one of the substrates of these enzymes. However, drugs that are simple competitive inhibitors will have to compete with the high concentrations of ATP in the cell. Protein kinases can also be inhibited by competition at the binding sites where the kinases interact with their substrate proteins, and most proteins are present inside cells at concentrations much lower than the concentration of ATP. As a consequence, if two protein kinase inhibitors both bind in the active site with similar affinity, but only one has to compete with ATP, then the competitive inhibitor at the protein-binding site will inhibit the enzyme more effectively. [24]

Types of irreversible inhibition (covalent inactivation) Edit

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 nucleophilic groups react with amino acid side chains to form covalent adducts. The residues modified are those with side chains containing nucleophiles such as hydroxyl or sulfhydryl groups these include the amino acids serine (as in DFP, right), cysteine, threonine, or tyrosine. [25]

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 hydrolyse the peptide bonds holding proteins together, releasing free amino acids. [26]

Irreversible inhibitors display time-dependent inhibition and their potency therefore cannot be characterised by an IC50 value. [1] [27] This is because the amount of active enzyme at a given concentration of irreversible inhibitor will be different depending on how long the inhibitor is pre-incubated with the enzyme. Instead, kobs/[I] values are used, [28] where kobs is the observed pseudo-first order rate of inactivation (obtained by plotting the log of % activity vs. time) and [I] is the concentration of inhibitor. The kobs/[I] parameter is valid as long as the inhibitor does not saturate binding with the enzyme (in which case kobs = kinact).

Analysis of irreversible inhibition Edit

As shown in the figure to the right, irreversible inhibitors have a short instance where they form a reversible non-covalent complex with the enzyme (EI or ESI) and this then reacts to produce the covalently modified "dead-end complex" EI* (an irreversible covalent complex). The rate at which EI* is formed is called the inactivation rate or kinact. Since formation of EI may compete with ES, binding of irreversible inhibitors can be prevented by competition either with substrate or with a second, reversible inhibitor. This protection effect is good evidence of a specific reaction of the irreversible inhibitor with the active site.

The binding and inactivation steps of this reaction are investigated by incubating the enzyme with inhibitor and assaying the amount of activity remaining over time. The activity will be decreased in a time-dependent manner, usually following exponential decay. Fitting these data to a rate equation gives the rate of inactivation at this concentration of inhibitor. This is done at several different concentrations of inhibitor. If a reversible EI complex is involved the inactivation rate will be saturable and fitting this curve will give kinact and Ki. [29]

Another method that is widely used in these analyses is mass spectrometry. Here, accurate measurement of the mass of the unmodified native enzyme and the inactivated enzyme gives the increase in mass caused by reaction with the inhibitor and shows the stoichiometry of the reaction. [30] This is usually done using a MALDI-TOF mass spectrometer. In a complementary technique, peptide mass fingerprinting involves digestion of the native and modified protein with a protease such as trypsin. This will produce a set of peptides that can be analysed using a mass spectrometer. The peptide that changes in mass after reaction with the inhibitor will be the one that contains the site of modification.

Special cases Edit

Not all irreversible inhibitors form covalent adducts with their enzyme targets. Some reversible inhibitors bind so tightly to their target enzyme that they are essentially irreversible. These tight-binding inhibitors may show kinetics similar to covalent irreversible inhibitors. In these cases, some of these inhibitors rapidly bind to the enzyme in a low-affinity EI complex and this then undergoes a slower rearrangement to a very tightly bound EI* complex (see figure above). This kinetic behaviour is called slow-binding. [32] This slow rearrangement after binding often involves a conformational change as the enzyme "clamps down" around the inhibitor molecule. Examples of slow-binding inhibitors include some important drugs, such methotrexate, [33] allopurinol, [34] and the activated form of acyclovir. [35]

Examples of irreversible inhibitors Edit

Diisopropylfluorophosphate (DFP) is shown as an example of an irreversible protease inhibitor in the figure above right. The enzyme hydrolyses the phosphorus–fluorine bond, but the phosphate residue remains bound to the serine in the active site, deactivating it. [36] Similarly, DFP also reacts with the active site of acetylcholine esterase in the synapses of neurons, and consequently is a potent neurotoxin, with a lethal dose of less than 100 mg. [37]

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, α-difluoromethylornithine or DFMO, which is an analogue of the amino acid ornithine, and is used to treat African trypanosomiasis (sleeping sickness). Ornithine decarboxylase can catalyse the decarboxylation of DFMO instead of ornithine, as shown above. However, this decarboxylation reaction is followed by the elimination of a fluorine atom, which converts this catalytic intermediate into a conjugated imine, a highly electrophilic species. This reactive form of DFMO then reacts with either a cysteine or lysine residue in the active site to irreversibly inactivate the enzyme. [31]

Since irreversible inhibition often involves the initial formation of a non-covalent EI complex, it is sometimes possible for an inhibitor to bind to an enzyme in more than one way. For example, in the figure showing trypanothione reductase from the human protozoan parasite Trypanosoma cruzi, two molecules of an inhibitor called quinacrine mustard are bound in its active site. The top molecule is bound reversibly, but the lower one is bound covalently as it has reacted with an amino acid residue through its nitrogen mustard group. [38]

New drugs are the products of a long drug development process, the first step of which is often the discovery of a new enzyme inhibitor. In the past the only way to discover these new inhibitors was by trial and error: screening huge libraries of compounds against a target enzyme and hoping that some useful leads would emerge. This brute force approach is still successful and has even been extended by combinatorial chemistry approaches that quickly produce large numbers of novel compounds and high-throughput screening technology to rapidly screen these huge chemical libraries for useful inhibitors. [39]

More recently, an alternative approach has been applied: rational drug design uses the three-dimensional structure of an enzyme's active site to predict which molecules might be inhibitors. [40] These predictions are then tested and one of these tested compounds may be a novel inhibitor. This new inhibitor is then used to try to obtain a structure of the enzyme in an inhibitor/enzyme complex to show how the molecule is binding to the active site, allowing changes to be made to the inhibitor to try to optimise binding. This test and improve cycle is then repeated until a sufficiently potent inhibitor is produced. [41] Computer-based methods of predicting the affinity of an inhibitor for an enzyme are also being developed, such as molecular docking [42] and molecular mechanics.

Enzyme inhibitors are found in nature and are also designed and produced as part of pharmacology and biochemistry. Natural poisons are often enzyme inhibitors that have evolved to defend a plant or animal against predators. These natural toxins include some of the most poisonous compounds known. Artificial inhibitors are often used as drugs, but can also be insecticides such as malathion, herbicides such as glyphosate, or disinfectants such as triclosan. Other artificial enzyme inhibitors block acetylcholinesterase, an enzyme which breaks down acetylcholine, and are used as nerve agents in chemical warfare.

Chemotherapy Edit

The most common uses for enzyme inhibitors are as drugs to treat disease. Many of these inhibitors target a human enzyme and aim to correct a pathological condition. However, not all drugs are enzyme inhibitors. Some, such as anti-epileptic drugs, alter enzyme activity by causing more or less of the enzyme to be produced. These effects are called enzyme induction and inhibition and are alterations in gene expression, which is unrelated to the type of enzyme inhibition discussed here. Other drugs interact with cellular targets that are not enzymes, such as ion channels or membrane receptors.

An example of a medicinal enzyme inhibitor is sildenafil (Viagra), a common treatment for male erectile dysfunction. This compound is a potent inhibitor of cGMP specific phosphodiesterase type 5, the enzyme that degrades the signalling molecule cyclic guanosine monophosphate. [43] This signalling molecule triggers smooth muscle relaxation and allows blood flow into the corpus cavernosum, which causes an erection. Since the drug decreases the activity of the enzyme that halts the signal, it makes this signal last for a longer period of time.

Another example of the structural similarity of some inhibitors to the substrates of the enzymes they target is seen in the figure comparing the drug methotrexate to folic acid. Folic acid is a substrate of dihydrofolate reductase, an enzyme involved in making nucleotides that is potently inhibited by methotrexate. Methotrexate blocks the action of dihydrofolate reductase and thereby halts the production of nucleotides. This block of nucleotide biosynthesis is more toxic to rapidly growing cells than non-dividing cells, since a rapidly growing cell has to carry out DNA replication, therefore methotrexate is often used in cancer chemotherapy. [44]

Antibiotics Edit

Drugs also are used to inhibit enzymes needed for the survival of pathogens. For example, bacteria are surrounded by a thick cell wall made of a net-like polymer called peptidoglycan. Many antibiotics such as penicillin and vancomycin inhibit the enzymes that produce and then cross-link the strands of this polymer together. [45] This causes the cell wall to lose strength and the bacteria to burst. In the figure, a molecule of penicillin (shown in a ball-and-stick form) is shown bound to its target, the transpeptidase from the bacteria Streptomyces R61 (the protein is shown as a ribbon-diagram).

Antibiotic drug design is facilitated when an enzyme that is essential to the pathogen's survival is absent or very different in humans. In the example above, humans do not make peptidoglycan, therefore inhibitors of this process are selectively toxic to bacteria. Selective toxicity is also produced in antibiotics by exploiting differences in the structure of the ribosomes in bacteria, or how they make fatty acids.

Metabolic control Edit

Enzyme inhibitors are also important in metabolic control. Many metabolic pathways in the cell are inhibited by metabolites that control enzyme activity through allosteric regulation or substrate inhibition. A good example is the allosteric regulation of the glycolytic pathway. This catabolic pathway consumes glucose and produces ATP, NADH and pyruvate. A key step for the regulation of glycolysis is an early reaction in the pathway catalysed by phosphofructokinase-1 (PFK1). When ATP levels rise, ATP binds an allosteric site in PFK1 to decrease the rate of the enzyme reaction glycolysis is inhibited and ATP production falls. This negative feedback control helps maintain a steady concentration of ATP in the cell. However, metabolic pathways are not just regulated through inhibition since enzyme activation is equally important. With respect to PFK1, fructose 2,6-bisphosphate and ADP are examples of metabolites that are allosteric activators. [46]

Physiological enzyme inhibition can also be produced by specific protein inhibitors. This mechanism occurs in the pancreas, which synthesises many digestive precursor enzymes known as zymogens. Many of these are activated by the trypsin protease, so it is important to inhibit the activity of trypsin in the pancreas to prevent the organ from digesting itself. One way in which the activity of trypsin is controlled is the production of a specific and potent trypsin inhibitor protein in the pancreas. This inhibitor binds tightly to trypsin, preventing the trypsin activity that would otherwise be detrimental to the organ. [47] Although the trypsin inhibitor is a protein, it avoids being hydrolysed as a substrate by the protease by excluding water from trypsin's active site and destabilising the transition state. [48] Other examples of physiological enzyme inhibitor proteins include the barstar inhibitor of the bacterial ribonuclease barnase. [49]

Pesticides Edit

Many pesticides are enzyme inhibitors. Acetylcholinesterase (AChE) is an enzyme found in animals, from insects to humans. It is essential to nerve cell function through its mechanism of breaking down the neurotransmitter acetylcholine into its constituents, acetate and choline. This is somewhat unusual among neurotransmitters as most, including serotonin, dopamine, and norepinephrine, are absorbed from the synaptic cleft rather than cleaved. A large number of AChE inhibitors are used in both medicine and agriculture. Reversible competitive inhibitors, such as edrophonium, physostigmine, and neostigmine, are used in the treatment of myasthenia gravis and in anaesthesia. The carbamate pesticides are also examples of reversible AChE inhibitors. The organophosphate pesticides such as malathion, parathion, and chlorpyrifos irreversibly inhibit acetylcholinesterase.

The herbicide glyphosate is an inhibitor of 3-phosphoshikimate 1-carboxyvinyltransferase, [50] other herbicides, such as the sulfonylureas inhibit the enzyme acetolactate synthase. Both these enzymes are needed for plants to make branched-chain amino acids. Many other enzymes are inhibited by herbicides, including enzymes needed for the biosynthesis of lipids and carotenoids and the processes of photosynthesis and oxidative phosphorylation. [51]

Natural poisons Edit

Animals and plants have evolved to synthesise a vast array of poisonous products including secondary metabolites, peptides and proteins that can act as inhibitors. Natural toxins are usually small organic molecules and are so diverse that there are probably natural inhibitors for most metabolic processes. [52] The metabolic processes targeted by natural poisons encompass more than enzymes in metabolic pathways and can also include the inhibition of receptor, channel and structural protein functions in a cell. For example, paclitaxel (taxol), an organic molecule found in the Pacific yew tree, binds tightly to tubulin dimers and inhibits their assembly into microtubules in the cytoskeleton. [53]

Many natural poisons act as neurotoxins that can cause paralysis leading to death and have functions for defence against predators or in hunting and capturing prey. Some of these natural inhibitors, despite their toxic attributes, are valuable for therapeutic uses at lower doses. [54] An example of a neurotoxin are the glycoalkaloids, from the plant species in the family Solanaceae (includes potato, tomato and eggplant), that are acetylcholinesterase inhibitors. Inhibition of this enzyme causes an uncontrolled increase in the acetylcholine neurotransmitter, muscular paralysis and then death. Neurotoxicity can also result from the inhibition of receptors for example, atropine from deadly nightshade (Atropa belladonna) that functions as a competitive antagonist of the muscarinic acetylcholine receptors. [55]

Although many natural toxins are secondary metabolites, these poisons also include peptides and proteins. An example of a toxic peptide is alpha-amanitin, which is found in relatives of the death cap mushroom. This is a potent enzyme inhibitor, in this case preventing the RNA polymerase II enzyme from transcribing DNA. [56] The algal toxin microcystin is also a peptide and is an inhibitor of protein phosphatases. [57] This toxin can contaminate water supplies after algal blooms and is a known carcinogen that can also cause acute liver hemorrhage and death at higher doses. [58]

Proteins can also be natural poisons or antinutrients, such as the trypsin inhibitors (discussed above) that are found in some legumes, as shown in the figure above. A less common class of toxins are toxic enzymes: these act as irreversible inhibitors of their target enzymes and work by chemically modifying their substrate enzymes. An example is ricin, an extremely potent protein toxin found in castor oil beans. This enzyme is a glycosidase that inactivates ribosomes. Since ricin is a catalytic irreversible inhibitor, this allows just a single molecule of ricin to kill a cell. [59]


Mechanism of Transformation (With Diagram) | Genetics

Notani and Setlow (1974) have described the mechanism of bacterial transformation. Moreover, in S. pneumoniae the competent state is transient and persists only for a short period. The competent state is induced by the competence activator protein of molecular weight of 1,000 Dalton.

It binds to the plasma membrane of receptor and triggers the synthesis of 10 new proteins within 10 minutes. The competence factor (CF) accelerates the process of transport or leakage of autolysin molecules into the periplasmic space. Moreover, in H. influenzae no competence factors have been reported.

Only Changes in cell envelope accompany the development of competence state. The cell envelope of competent cells contains increased level of polysacccharide as compared to the cells of log phase.

Structural changes in competent cells induce numerous vesicles called transformosome buds on the surface that contains protein and mediates the uptake of transforming DNA. Transformation is accomplished in the following steps (Fig.8.4).

Fig. 8.4 : Diagrammatic presentation of transformation in streptococci.

As a result of random collision, DNA comes first in the contact of cell surface of competent bacteria (Figs. 8.4 and 8.5 A-B). First the DNA binding is reversible and lasts for about 4-5 seconds. Thereafter, it becomes irreversible permanently. For about 2 minutes it remains in non-transforming state. Thereafter, before 5 minutes it is converted into the transforming state.

The period (about 10 minutes) during which no transformation occurs in competent recipient cells is called eclipse. Both types of DNA, transforming and non-transforming, bind to the cell surface where the receptor sites are located. In B. subtilis membrane vesicles in competent cells are found that bind to 20 mg of dsDNA/mg of membrane protein. The competent cells show six fold more DNA binding sites than the non-competent cells.

In H influenzae transformosome bud forms the surface and contains proteins that mediate DNA uptake. It binds with conserved sequence (5’AAGTGCGGTCA 3′) present at 4 kb interval on DNA. The DNA uptake site contains two proteins of 28 and 52 kilo-Daltons. After binding, the receptor proteins present the donor DNA to the membrane associated uptake sites.

In S. pneumoniae the CF induces the ability to bind DNA molecules.

The DNA molecules that bind permanently enter the competent recipient cells. DNA is also resistant to DNase degradation. The nucleolytic enzymes located at the surface of competent recipient cells act upon the donor DNA molecule when it binds the cell membrane.

The endonuclease-1 of the recipient cells which is associated with cell membrane acts as DNA translocase by attacking and degrading one strand of the dsDNA. Consequently only complementary single strand of DNA enters into the recipient cells (Figs. 8.4 and 8.5A).

It has been confirmed by performing the experiments with radiolabelling of donor DNA. The mutant cells of S. pneumoniae lack endonuclease – 1, therefore, transformation does not occur. Interestingly in B. subtilis degradation of one strand is being delayed. Hence, both the strands enter the recipient cell. The upper limit of peneterating DNA into the recipient cell is about 750 base pairs.

The size of donor DNA affects transformation. Successful transformation occurs with the donor DN.A of molecular weight between 30,00,000 and 8 million Dalton. With increasing the concentration of donor DNA the number of competent cells increases. DNA uptake process is the energy requiring mechanism because it can be inhibited by the energy requiring inhibitors.

After penetration the donor DNA migrates from periphery of cell to the bacterial DNA. This movement in different bacteria differs. For example, in B. subtilis this movement occurs for about 16-60 minutes. During this movement, DNA is associated with mesosomes which possibly transport it to the bacterial DNA.

(c) Synapsis formation:

The single stranded DNA is coated with SSB proteins, which maintain, the single stranded region in a replication fork (Fig. 8.5B). The single strand of the donor DNA or portion of it is linearly inserted into the recipient DNA (Fig. 8.5 C-D). The bacterial protein like E. coli RecA protein probably facilitates the DNA pairing during recombination. It causes the local unwinding of dsDNA of the recipient cell from the 5′ end.

How the displaced single strand is cut, still not known? Base pairing i.e. synapsis occurs between the homologous donor ssDNA and the recipient DNA. Unwinding of the recipient DNA continues at the end of assimilated DNA and allows the fraction of invading DNA to increase base pairs. This process is called branch migration (F).

The endonuclease cuts the unpaired free end of donor DNA or the recipient DNA. This process is called trimming (Fig. 8.5E-F). The nick is sealed by DNA ligase (G). Consequently, a heteroduplex region containing a mismatched base pairs is formed (H). Furthermore, in the progenies whether the donor marker is or is not recovered, depends on the occurrence of mismatch repair.

If the mismatch repair occurs again, it depends whether the unpaired base in the donor or recipient strand is removed. After replication the heteroduplex forms the homo-duplexes, one of these is of normal type and the second is transformed duplex. The normal duplex is from the recipient cell in origin, whereas the transformed duplex is from the donor genome.

The efficiency of integration of genetic markers into the genome of recipient cell varies with different genes that the recipient cell possesses. This genetic trait is called hex (high efficiency of integration). The hex system eliminates a large fraction of low efficiency (LE) markers and permits high efficiency (HE) markers to be integrated. Therefore, the hex function is a mismatch-base correction system.

The donor genes differing from the recipient genes by a single base pair create a mismatch when integrated initially. The hex mismatch repair system (with LE markers) can correct either of donor strands. Therefore, there is fifty-fifty chance for a given marker to be retained. The HE markers correct only the recipient strand.

For the LE markers, hex mismatch repair system unusually removes the mismatched bases of the donor DNA and the cell retains the recipient genotype, whereas for HE markers the same system removes the mismatched bases of recipient DNA and the cell consists of donor genotype.

In the later case, after replication of chromosome and cell division the one progeny cell contains the donor genotype and the other has the recipient genotype. These two types of cells can be differentiated through plating method by using the antibiotic markers.

However, for pneumococci it is a general feature that all the strains discriminate between LH and HE markers when transformation has occurred with homologous DNA. The hex – cells (mutant in hex function) fail to discriminate between the two markers and, therefore, integrate all markers with high efficiency, because one of the two daughter cells after cell division contains the genotype.


Summary – Reversible vs Irreversible Inhibition

Enzyme inhibition can be either reversible or irreversible. In summarizing the difference between reversible and irreversible inhibition in reversible inhibition, the inhibitor binds with the enzyme non-covalently. Hence, the unbinding of the inhibitor from the enzyme is easy and rapid. On the other hand, in irreversible inhibition, the inhibitor binds with the enzyme covalently. Therefore, the inhibitor strongly binds with the enzyme and the dissociation of the enzyme-inhibitor complex is slow and hard. Therefore, this is the key difference between reversible and irreversible inhibition. Furthermore, in reversible inhibition, the reaction can be reversed, and the enzyme can be reactivated again. But in irreversible inhibition, the reaction cannot be reversed, and the enzyme cannot be activated again.

Reference:

1. “Enzyme Inhibitor.” Wikipedia, Wikimedia Foundation, 1 Jan. 2019. Available here
2. “Enzyme Inhibitor.” NeuroImage, Academic Press. Available here

Image Courtesy:

1.”DHFR methotrexate inhibitor”By Thomas Shafee – Own work, (CC BY 4.0) via Commons Wikimedia
2.”Covalent-drugs-silence-proteins”By Dr. Juswinder Singh (CC BY-SA 3.0) via Commons Wikimedia


5A: Introduction to Reversible Binding - Biology

There are two types of reversible inhibitors:

In both cases reversible inhibitors can be removed from the enzymes - but only under certain conditions.

Competitive inhibition

Competitive inhibitors are inhibitors that compete with substrates for the active site. They resemble the substrate in that they can fit into the active site,fooling the enzyme into thinking that they are substrates. They differ from the substrate in that they are unreactive. They therefore reduce the number of enzymes available to catalyse a reaction. If there is enough substrate available though, the chances of an enzyme attracting an inhibitor is smaller - most enzymes will still attract the correct substrates and a reaction can still occur. To help overcome the effect of a competitive inhibitor, the substrate concentration should be increased. The inhibitors usually leave the active site when the substrate concentration is high enough.


Inhibitors are not always poisons or harmful. An example of an inhibitor used in treating poisons is in poisoning. Ethylene glycol is a component of car antifreezes. By itself it is a harmless substance but it is broken down in the body into oxalic acid (a deadly poison) by the enzyme, alcohol dehydrogenase. Alcohol (ethanol) acts as a competitive inhibitor for alcohol dehydrogenase. Giving the patient large amounts of alcohol will cause the ethanol to compete with ethylene glycol for the active site of alcohol dehydrogenase. Alcohol is the preferred substrate for alcohol dehydrogenase so when it is present, it binds with the enzyme. After a while, the ethylene glycol is harmlessly excreted.

Various poisons such as cyanide, arsenic and mercury - in fact many heavy metal ions - act as inhibitors. HCN binds to the active site of various cytochromases which are enzymes that are responsible for catalysing oxidation and reduction processes in cellular respiration. Many diet pills of the 1920s contained arsenic! These inhibited certain digestive enzymes in the human alimentary canal.

Non-competitive inhibition

These substances do not bind to the active site of an enzyme, but rather to other parts of the enzyme. In doing so, they may change the conformation of the active site as has already been explained (breaking certain hydrogen bonds and forming incorrect ones, changing the shape of the active site) and possibly inactivate it.

A non-competitive inhibitor will eventually leave the binding site. Substrate molecules have nothing to do with this, as these inhibitors do not bind to the active site, and therefore have a greater inhibitory effect. A greater substrate concentration will not help as substrate molecules only compete for positions at the active site. Examples include lead, mercury and chromium.


Many pesticides and herbicides are also inhibitors. Read the pamphlet inside the boxes of garden or household poisons and see how these function. Look at medicine pamphlets as well. You will often find that many use inhibitors that block active sites of bacterial enzymes, killing them or stopping their reproduction. Some painkillers also act as inhibitors, eg aspirin.

Explanation

When a competitive inhibitor is present, the enzyme activity is decreased compared to when no inhibitor is present. As the substrate concentration is increased, the activity eventually becomes the same.

You will notice that the optimum substrate concentration is much higher.

With a non-competitive inhibitor, the inhibition is not dependent on the substrate concentration and the effect is similar to an irreversible inhibitor.


Reversible Reactions

In reversible reactions, the reactants and products are never fully consumed they are each constantly reacting and being produced. A reversible reaction can take the following summarized form:

This reversible reaction can be broken into two reactions.

Reaction 1: [ A + B xrightarrowC+D ]

Reaction 2: [ C + D xrightarrow>A+B ]

These two reactions are occurring simultaneously , which means that the reactants are reacting to yield the products, as the products are reacting to produce the reactants . Collisions of the reacting molecules cause chemical reactions in a closed system. After products are formed, the bonds between these products are broken when the molecules collide with each other, producing sufficient energy needed to break the bonds of the product and reactant molecules.

Below is an example of the summarized form of a reversible reaction and a breakdown of the reversible reaction N2O4 &harr 2NO2

Reaction 1 and Reaction 2 happen at the same time because they are in a closed system.

Blue : Nitrogen Red : Oxygen

Imagine a ballroom. Let reactant A be 10 girls and reactant B be 10 boys. As each girl and boy goes to the dance floor, they pair up to become a product. Once five girls and five boys are on the dance floor, one of the five pairs breaks up and moves to the sidelines, becoming reactants again. As this pair leaves the dance floor, another boy and girl on the sidelines pair up to form a product once more. This process continues over and over again, representing a reversible reaction.

Unlike irreversible reactions, reversible reactions lead to equilibrium: in reversible reactions, the reaction proceeds in both directions whereas in irreversible reactions the reaction proceeds in only one direction. To learn more about this phenomenon, click here: Chemical Equilibrium

If the reactants are formed at the same rate as the products, a dynamic equilibrium exists. For example, if a water tank is being filled with water at the same rate as water is leaving the tank (through a hypothetical hole), the amount of water remaining in the tank remains consistent.


Reversible covalent direct thrombin inhibitors

Introduction: In recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high.

Methods: We have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1.

Results: We demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay.

Conclusion: Compound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles.

Conflict of interest statement

MS, MR, KL, TPS, DMC, LI, SC, PZ, MAE, KMS, DCW, AD, and DBK are employees of Verseon Corporation. EDC discloses a financial interest in Verseon Corporation and funding through NIH grants HL049413, HL073813, and HL112303. NP discloses a financial interest in Hemadvance, LLC and funding through AHA grant AHA15SDG25550094. KMS and DCW are inventors on a patent application (WO/2014/149139) that includes Compound 1 and has local applications pending in numerous jurisdictions worldwide. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Structures of Compound 1, the…

Fig 1. Structures of Compound 1, the dansylated Compound 2, and the deacylated Compound 3…

Fig 2. Interaction of Compound 1 with…

Fig 2. Interaction of Compound 1 with thrombin.

Compound 1 at 1 μM was incubated…

Fig 4. Structural model of the thrombin…

Fig 4. Structural model of the thrombin active site.

The structural model of the thrombin…

Fig 5. Interaction diagram for the thrombin…

Fig 5. Interaction diagram for the thrombin active site modified by Compound 1.

Fig 6. Kinetic characterization of Compound 1.

Fig 6. Kinetic characterization of Compound 1.

Fig 7. Spontaneous recovery of thrombin activity.

Fig 7. Spontaneous recovery of thrombin activity.

% Thrombin activity was recorded as a function…

Fig 8. Thrombograms from the thrombin generation…

Fig 8. Thrombograms from the thrombin generation assay for argatroban, dabigatran, and Compound 1.


AFFINITY CHROMATOGRAPHY OF MINIMAL AMOUNT OF ACETYLCHOLINESTERASE FROM NERVOUS TISSUES AND ERYTHROCYTE MEMBRANES

Publisher Summary

This chapter discusses the affinity chromatography of minimal amount of acetylcholinesterase from nervous tissues and erythrocyte membranes. It discusses a study in which a suitable method for the selective isolation of AChE from other proteins, by affinity chromatography, was worked out by integrating and modifying three available methods. Affigel 202 (Bio-Rad) was covalently linked to a specific and reversible AChE inhibitor. This inhibitor was synthesized, and its properties as the differential inhibitor of AChE and butyrilcholinesterase (3.1.1.8.) (BuChE) were examined. The values of PI 50 were found to be 3.8, 4.0, and 2.0 for rat brain AChE, electric eel AChE, and horse serum BuChE, respectively. According to the batchwise procedure, finally adopted, the sample containing at least 60 mU was loaded onto a similar volume of the affinity chromatography gel and the unbound proteins were removed by centrifugation. After three washings with a threefold volume of saline phosphate buffer, AChE was eluted in two steps from the gel by choline chloride solution, which proved effective because of the poor affinity of the linked inhibitor for AChE. The AChE activity, recovered after centrifugation and dialysis of the supernatant, was about 90% of the activity in the sample.

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