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Learning Objectives
- Explain what an enzyme inhibitor is.
- Distinguish between reversible and irreversible inhibitors.
- Distinguish between competitive and noncompetitive inhibitors.
Previously, we noted that enzymes are inactivated at high temperatures and by changes in pH. These are nonspecific factors that would inactivate any enzyme. The activity of enzymes can also be regulated by more specific inhibitors. Many compounds are poisons because they bind covalently to particular enzymes or kinds of enzymes and inactivate them (Table \(\PageIndex{1}\)).
| Poison | Formula | Example of Enzyme Inhibited | Action |
|---|---|---|---|
| arsenate | \(\ce{AsO4^{3−}}\) | glyceraldehyde 3-phosphate dehydrogenase | substitutes for phosphate |
| iodoacetate | \(\ce{ICH2COO^{−}}\) | triose phosphate dehydrogenase | binds to cysteine \(\ce{SH}\) group |
| diisopropylfluoro-phosphate (DIFP; a nerve poison) |  | acetylcholinesterase | binds to serine \(\ce{OH}\) group |
Irreversible Inhibition: Poisons
An irreversible inhibitor inactivates an enzyme by bonding covalently to a particular group at the active site. The inhibitor-enzyme bond is so strong that the inhibition cannot be reversed by the addition of excess substrate. The nerve gases, especially Diisopropyl fluorophosphate (DIFP), irreversibly inhibit biological systems by forming an enzyme-inhibitor complex with a specific OH group of serine situated at the active sites of certain enzymes. The peptidases trypsin and chymotrypsin contain serine groups at the active site and are inhibited by DIFP.
Reversible Inhibition
A reversible inhibitor inactivates an enzyme through noncovalent, more easily reversed, interactions. Unlike an irreversible inhibitor, a reversible inhibitor can dissociate from the enzyme. Reversible inhibitors include competitive inhibitors and noncompetitive inhibitors. (There are additional types of reversible inhibitors.) A competitive inhibitor is any compound that bears a structural resemblance to a particular substrate and thus competes with that substrate for binding at the active site of an enzyme. The inhibitor is not acted on by the enzyme but does prevent the substrate from approaching the active site.
The degree to which a competitive inhibitor interferes with an enzyme’s activity depends on the relative concentrations of the substrate and the inhibitor. If the inhibitor is present in relatively large quantities, it will initially block most of the active sites. But because the binding is reversible, some substrate molecules will eventually bind to the active site and be converted to product. Increasing the substrate concentration promotes displacement of the inhibitor from the active site. Competitive inhibition can be completely reversed by adding substrate so that it reaches a much higher concentration than that of the inhibitor.
Studies of competitive inhibition have provided helpful information about certain enzyme-substrate complexes and the interactions of specific groups at the active sites. As a result, pharmaceutical companies have synthesized drugs that competitively inhibit metabolic processes in bacteria and certain cancer cells. Many drugs are competitive inhibitors of specific enzymes.
A classic example of competitive inhibition is the effect of malonate on the enzyme activity of succinate dehydrogenase (Figure \(\PageIndex{1}\)). Malonate and succinate are the anions of dicarboxylic acids and contain three and four carbon atoms, respectively. The malonate molecule binds to the active site because the spacing of its carboxyl groups is not greatly different from that of succinate. However, no catalytic reaction occurs because malonate does not have a CH2CH2 group to convert to CH=CH. This reaction will also be discussed in connection with the Krebs cycle and energy production.
To Your Health: Penicillin
Chemotherapy is the strategic use of chemicals (that is, drugs) to destroy infectious microorganisms or cancer cells without causing excessive damage to the other, healthy cells of the host. From bacteria to humans, the metabolic pathways of all living organisms are quite similar, so the search for safe and effective chemotherapeutic agents is a formidable task. Many well-established chemotherapeutic drugs function by inhibiting a critical enzyme in the cells of the invading organism.
An antibiotic is a compound that kills bacteria; it may come from a natural source such as molds or be synthesized with a structure analogous to a naturally occurring antibacterial compound. Antibiotics constitute no well-defined class of chemically related substances, but many of them work by effectively inhibiting a variety of enzymes essential to bacterial growth.
Penicillin, one of the most widely used antibiotics in the world, was fortuitously discovered by Alexander Fleming in 1928, when he noticed antibacterial properties in a mold growing on a bacterial culture plate. In 1938, Ernst Chain and Howard Florey began an intensive effort to isolate penicillin from the mold and study its properties. The large quantities of penicillin needed for this research became available through development of a corn-based nutrient medium that the mold loved and through the discovery of a higher-yielding strain of mold at a United States Department of Agriculture research center near Peoria, Illinois. Even so, it was not until 1944 that large quantities of penicillin were being produced and made available for the treatment of bacterial infections.
Penicillin functions by interfering with the synthesis of cell walls of reproducing bacteria. It does so by inhibiting an enzyme—transpeptidase—that catalyzes the last step in bacterial cell-wall biosynthesis. The defective walls cause bacterial cells to burst. Human cells are not affected because they have cell membranes, not cell walls.
Several naturally occurring penicillins have been isolated. They are distinguished by different R groups connected to a common structure: a four-member cyclic amide (called a lactam ring) fused to a five-member ring. The addition of appropriate organic compounds to the culture medium leads to the production of the different kinds of penicillin.
The penicillins are effective against gram-positive bacteria (bacteria capable of being stained by Gram’s stain) and a few gram-negative bacteria (including the intestinal bacterium Escherichia coli). They are effective in the treatment of diphtheria, gonorrhea, pneumonia, syphilis, many pus infections, and certain types of boils. Penicillin G was the earliest penicillin to be used on a wide scale. However, it cannot be administered orally because it is quite unstable; the acidic pH of the stomach converts it to an inactive derivative. The major oral penicillins—penicillin V, ampicillin, and amoxicillin—on the other hand, are acid stable.
Some strains of bacteria become resistant to penicillin through a mutation that allows them to synthesize an enzyme—penicillinase—that breaks the antibiotic down (by cleavage of the amide linkage in the lactam ring). To combat these strains, scientists have synthesized penicillin analogs (such as methicillin) that are not inactivated by penicillinase.
Some people (perhaps 5% of the population) are allergic to penicillin and therefore must be treated with other antibiotics. Their allergic reaction can be so severe that a fatal coma may occur if penicillin is inadvertently administered to them. Fortunately, several other antibiotics have been discovered. Most, including aureomycin and streptomycin, are the products of microbial synthesis. Others, such as the semisynthetic penicillins and tetracyclines, are made by chemical modifications of antibiotics; and some, like chloramphenicol, are manufactured entirely by chemical synthesis. They are as effective as penicillin in destroying infectious microorganisms. Many of these antibiotics exert their effects by blocking protein synthesis in microorganisms.
Initially, antibiotics were considered miracle drugs, substantially reducing the number of deaths from blood poisoning, pneumonia, and other infectious diseases. Some seven decades ago, a person with a major infection almost always died. Today, such deaths are rare. Seven decades ago, pneumonia was a dreaded killer of people of all ages. Today, it kills only the very old or those ill from other causes. Antibiotics have indeed worked miracles in our time, but even miracle drugs have limitations. Not long after the drugs were first used, disease organisms began to develop strains resistant to them. In a race to stay ahead of resistant bacterial strains, scientists continue to seek new antibiotics. The penicillins have now been partially displaced by related compounds, such as the cephalosporins and vancomycin. Unfortunately, some strains of bacteria have already shown resistance to these antibiotics.
Some reversible inhibitors are noncompetitive. A noncompetitive inhibitor can combine with either the free enzyme or the enzyme-substrate complex because its binding site on the enzyme is distinct from the active site. Binding of this kind of inhibitor alters the three-dimensional conformation of the enzyme, changing the configuration of the active site with one of two results. Either the enzyme-substrate complex does not form at its normal rate, or, once formed, it does not yield products at the normal rate. Because the inhibitor does not structurally resemble the substrate, the addition of excess substrate does not reverse the inhibitory effect.
Feedback inhibition is a normal biochemical process that makes use of noncompetitive inhibitors to control some enzymatic activity. In this process, the final product inhibits the enzyme that catalyzes the first step in a series of reactions. Feedback inhibition is used to regulate the synthesis of many amino acids. For example, bacteria synthesize isoleucine from threonine in a series of five enzyme-catalyzed steps. As the concentration of isoleucine increases, some of it binds as a noncompetitive inhibitor to the first enzyme of the series (threonine deaminase), thus bringing about a decrease in the amount of isoleucine being formed (Figure \(\PageIndex{2}\)).
Summary
An irreversible inhibitor inactivates an enzyme by bonding covalently to a particular group at the active site. A reversible inhibitor inactivates an enzyme through noncovalent, reversible interactions. A competitive inhibitor competes with the substrate for binding at the active site of the enzyme. A noncompetitive inhibitor binds at a site distinct from the active site.
FAQs
How do you calculate enzyme inhibition? ›
The Michaelis-Menten equation with a competitive inhibitor present: vo = Vmax[S]/(aKM + [S] ) , where.
What results from enzyme inhibition? ›Enzyme inhibition can be used to prolong the effects of endocannabinoids in the reduction of IOP. Enzyme inhibition works indirectly to regulate IOP: by inhibiting degradation enzymes, endocannabinoids are no longer degraded, and free to work on receptors.
What are the 3 types of enzyme inhibitions? ›Enzyme inhibition is an important means of regulating activity in living cells. There are three basic types of enzyme inhibition: competitive, noncompetitive, and uncompetitive.
How do you know if an inhibitor is competitive or noncompetitive? ›The decrease in Vmax and the unchanged Km is the primary way to differentiate noncompetitive inhibition from competitive (no direct change in Vmax, increased Km) and uncompetitive (decreased Vmax and Km).
How do you calculate the percentage of inhibition? ›Percentage of inhibition: (Control OD − (Sample OD/Control OD)) × 100.
How do you calculate 50% inhibition? ›IC50 is always one value and it is the concentration which shows 50% inhibition. It is calculated from the plot of serial dilutions vs the % inhibition. IC50 = (Conc. of tested agent × 50)/% inhibition.
What is degree of inhibition? ›Because the degree of inhibition is a ratio between rates, kinetic data are normalized by the introduction of an internal control-the rate of the uninhibited reaction. Therefore, the error associated with the kinetic measurements decreases and less experimental measurements are necessary to achieve the diagnosis.
What are the 2 types of enzyme inhibition? ›Explanation: The molecule in the question is classified as an enzyme inhibitor because it inhibits an enzymatic reaction. There are two types of inhibitors; competitive and noncompetitive inhibitors.
What is an enzyme inhibition? ›A substance that blocks the action of an enzyme. Enzymes help speed up chemical reactions in the body and take part in many cell functions, including cell signaling, growth, and division. In cancer treatment, enzyme inhibitors may be used to block certain enzymes that cancer cells need to grow.
What is a high Ki value? ›Inhibitors with Ki values less than 100 μM were considered potent inhibitors. Inhibitors with Ki values higher than 100 μM were considered nonpotent inhibitors.
How do you measure the rate of an enzymatic reaction? ›
The reaction rate can therefore be measured with a colorimeter, which will indicate the absorbance of light through the product. The spectrophotometer shown below is similar to a colorimeter, although it measures the transmission, rather than the absorbtion of light.
What are enzyme 11 inhibitors? ›There are certain molecules(inhibitors) which interfere with the enzyme activity and does not lead to the formation of the product. This is known as enzyme inhibition. These inhibitors can bind to the active sites and prevent/interfere with the further activity.
How do you determine inhibition type? ›We can identify the type of reversible inhibition by observing how a change in the inhibitor's concentration affects the relationship between the rate of reaction and the substrate's concentration.
How does an inhibitor affect reaction rate? ›Inhibitors are molecules that prevent the action of catalysts. They bind to catalysts and prevent substrate binding, thereby halting the catalytic action. Since catalysts increase the speed of a reaction, addition of an inhibitor will lower the speed of the reaction.
What makes an inhibitor competitive? ›Competitive inhibition occurs when molecules very similar to the substrate molecules bind to the active site and prevent binding of the actual substrate. Penicillin, for example, is a competitive inhibitor that blocks the active site of an enzyme that many bacteria use to construct their cell…
What is 50% inhibitory dilution? ›The half-maximal inhibitory concentration (IC50) is the most widely used and informative measure of a drug's efficacy. It indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of potency of an antagonist drug in pharmacological research.
Can you have a negative percent inhibition? ›It is highly unlikely to obtain negative %inhibition in DPPH assay, unless you followed a wrong protocol/formula.
How do you calculate growth rate inhibition? ›k(c) = the average growth-rate of treated cells throughout the experiment. GR(c) = 2^[ k(c)/k(0) ] - 1 = the growth-rate inhibition value (GR value) of a given treatment at concentration c.
Which IC50 value is good? ›In most cases, the IC50 of the best candidate compound should be lower to 10 micromolar, acceptable for NIH to screen the NCI60 program. In addition, the drug design should think over the related problem about the absorption, distribution, metabolism and excretion in our body.
What is inhibition percentage IC50? ›The IC50 is the concentration of drug required for 50% inhibition. IC50 is an operational term dependent on the assay conditions. IC90 or IC99 is sometimes used when complete inhibition is required.
What is a low inhibition constant? ›
The LOWER the Ki for a particular drug at a particular receptor, the STRONGER its binding affinity for that receptor. This is because the lower Ki means that the drug can occupy 50% of those receptors even when the drug is present in a lower concentration.
How do you determine the inhibition constant? ›The inhibition constant Ki in the common case of competitive inhibition can be obtained by simple comparison of progress curves in the presence and in the absence of inhibitor. The difference between the times taken for the concentration of substrate to fall to the same value is used to obtain Ki.
How do you calculate inhibitor constant? ›The dissociation constant for the inhibitor is KI = [E][I]/[EI]. Steady-state analysis of the effect of the inhibitor shows that KM is increased by a factor of (1 + [I]/KI).
What are inhibitors Class 12? ›Inhibitors are chemical compounds that slow down the reaction. They sometimes even stop the reaction. They work exactly opposite of catalysts. Catalyst and Inhibitors. Catalyst enhances the rate of reaction without participating in the reaction, whereas Inhibitors stop or slow down the reaction.
How does pH affect enzyme activity? ›Enzyme activity is at its maximum value at the optimum pH. As the pH value is increased above or decreased below the optimum pH the enzyme activity decreases.
What is enzyme inhibition classify? ›Enzyme inhibitors can be classified into 2 separate categories according to their mode of attachment on the active site of the enzymes. (i) Competitive inhibitors. (ii) non-competitive inhibitors. Inhibition of enzymatic activity by enzyme inhibitors. (i) Competitive inhibitors.
Are enzyme inhibitors good or bad? ›It is an essential way of maintaining homeostasis in the cell. Cellular inhibitors can also be proteins which have selective binding and only bind to their target enzyme. This is important in aiding to control the enzymes that damage the cell, for example, nucleases and proteases.
What drugs cause enzyme inhibition? ›| Drug | Drug Description |
|---|---|
| Sildenafil | A phosphodiesterase inhibitor used for the treatment of erectile dysfunction. |
| Pyrimethamine | An antiparasitic drug used in the prevention and treatment of toxoplasmosis and malaria. |
| Remikiren | For the treatment of hypertension and heart failure |
A shy child at a birthday party might have fun only after abandoning her inhibitions and joining a game of musical chairs. An inhibition is a force that prevents something from happening—and often comes from you yourself. Shy people are often said to suffer from inhibitions.
Does high Ki mean better inhibitor? ›The smaller the Ki the greater the binding affinity and the smaller the amount of ligand is needed to inhibit its binding partners activity.
How do you calculate KI? ›
For competitive and uncompetitive inhibitors when the assay conditions are [S] = Km, then Ki = I50/2. For different conditions of [S] there is a divergence between competitive and uncompetitive inhibitors that may be used to identify the type of inhibitor. The equation for Ki also differs.
What does higher Ki mean? ›If the complex tends to fall apart easily, (high Ki) the enzyme will be free to function more normally. i.e. if the Ki is high, the inhibitory effect will be weak. A small Ki means that the inhibitor is bound tightly, and the amount of active enzyme present will be small so the inhibitory effect will be strong.
What are enzymatic rates? ›The rate of enzyme reaction is measured by the amount of substrate changed or amount of product formed during a period of time. The rate is determined by measuring the slope of the tangent to the curve in the initial stage of the reaction. The steeper the slope, the greater is the rate.
How do you calculate enzyme activity? ›Specific enzyme activity (usually stated simply as 'specific activity') is the number of enzyme units per ml divided by the concentration of protein in mg/ml. Specific activity values are therefore quoted as units/mg or nmol/min/mg (if unit definition B is applied).
What is the maximum rate of reaction? ›The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction, Vmax. The relationship between rate of reaction and concentration of substrate depends on the affinity of the enzyme for its substrate.
What diseases are enzyme inhibitors? ›- Hepatosplenomegaly (abnormally enlarged liver and/or spleen)
- Anemia (low levels of circulating red blood cells)
- Thrombocytopenia (low levels of platelets)
- Easy bruising.
- Lung disease.
- Bone abnormalities such as bone pain, fractures, and arthritis (swelling and pain in joints)
(eer-ree-VER-sih-bul EN-zime in-HIH-bih-ter) A substance that permanently blocks the action of an enzyme. In cancer treatment, irreversible enzyme inhibitors may block certain enzymes that cancer cells need to grow and may kill cancer cells. They are being studied in the treatment of some types of cancer.
Where are enzyme inhibitors found? ›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.
How do you calculate inhibition constant? ›The dissociation constant for the inhibitor is KI = [E][I]/[EI]. Steady-state analysis of the effect of the inhibitor shows that KM is increased by a factor of (1 + [I]/KI).
How do you calculate inhibition assay? ›It is typically calculated by the number of enzyme units per mL divided by the protein concentration of the enzyme in mg/mL and expressed as units/per mg protein.
What is the inhibition rate? ›
Known as: % INHIBITION, %{INHIBITION} The rate of measured normal activity minus inhibited activity, divided by the rate of normal activity of a given object.
What is inhibition rate of reaction? ›Inhibitors are molecules that prevent the action of catalysts. They bind to catalysts and prevent substrate binding, thereby halting the catalytic action. Since catalysts increase the speed of a reaction, addition of an inhibitor will lower the speed of the reaction.
How do you calculate inhibitor efficiency? ›Inhibitor Efficiency (%) = 100·(CRuninhibited- CRinhibited)/ CR. In general, the efficiency of an inhibitor increases with an increase in inhibitor concentration, e.g. a typically good inhibitor would give 95% inhibition at a concentration of 0.008% and 90% at a concentration of 0.004%.
Can percentage inhibition be negative? ›It is highly unlikely to obtain negative %inhibition in DPPH assay, unless you followed a wrong protocol/formula. You may recheck the following points and revisit the assay protocol: 1.
What is the optimal range for enzyme activity? ›Optimal ranges are evidence-based ranges that are associated with the lowest risk of disease and mortality. These can help overcome a lot of the issues with traditional reference ranges.
How do you calculate the enzyme assay? ›In enzyme assays, the Eadie–Scatchard equation is of the formv[S]=1Km×v+VmaxKmwhere v is the amount of product formed, [S] is the concentration of the substrate, Km is the Michaelis constant, and Vmax is the maximal theoretical velocity of the reaction.
How do you calculate enzyme concentration? ›For example, if the enzyme concentration is 0.147 µg/µl, and the specific activity is 152 pmol/ µg min, the enzyme concentration is: 0.147 µg/µl x 152 pmol/ µg min = 22 pmol/ µl min = 22 nmol/ min ml = 22 U/ml. It needs to be diluted 1: 22 to achieve 1U/ml.