Enzyme Inhibition By-Shubhani singh thakur
Description: Enzyme Inhibition By-Shubhani singh thakur Department of biochemistry Enzyme InhibitionInhibitor: An Enzyme inhibitor is a compound that decreases or tends to decrease the rate of an enzyme catalyzed reaction by influencing the binding of
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slide1. Enzyme Inhibition By-Shubhani singh thakur Department of biochemistry<br>
slide2. Enzyme Inhibition/Inhibitor: An Enzyme inhibitor is a compound that decreases or tends to decrease the rate of an enzyme catalyzed reaction by influencing the binding of substrate or its turnover number.
Blocking an enzyme's activity by chemical molecules which resemble or mimic a particular enzymes substrate(s) is called Enzyme Inhibition and the mole is said to be an Inhibitor. Therefore, many therapeutic drugs are some type of enzyme inhibitor.
Molecules that bind to enzymes and increase their activity are called Enzyme Activators. The binding of an inhibitor can stop a substrate from entering the enzyme's active site or hinder the enzyme from catalysing its reaction.
The modes and types of inhibitors have been classified by their kinetic activities and sites of actions.<br>
slide3. Type of Enzyme Inhibitors Reversible Irreversible Type of
Inhibitors Competitive Uncompetitive Non- Competitive Active Site
Directed Suicide
Inhibitors Mixed<br>
slide4. Types of Inhibition 1.Reversible Inhibition:Â
Reversible inhibitors bind non-covalently (hydrogen bonds, hydrophobic interactions and ionic bonds) and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.
Inhibitor binds to Enzyme reversibly through weak non- covelent interactions
An Equilibrium is established between the free inhibitor & EI Complex and is defined by an equilibrium constant (Ki)
The activity of Enzyme is fully restored on removing the inhibitor by dialysis.
Reversible inhibitors depending on concentration of E, S and I, show a definite degree of inhibition which is reached fairly rapidly and remains constant when initial velocity studies are carried out.<br>
slide5. 2.Irreversible Inhibition:Â
Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors modify key amino acid residues needed for enzymatic activity.
Inhibitor binds at or near the active site of the enzyme irreversibly, usually by covalent bonds, so it can’t dissociate from the enzyme
No equilibrium exits
Enzyme activity is not regained on dialysis
Effectiveness of inhibitor is expressed not by equilibrium constant but by a velocity constant, which determines the fraction of the enzyme inhibited in a given period of time by a certain concentration of the inhibitor<br>
slide6. A competitive inhibitor combines with the free enzyme to form an EI complex in a manner that prevents substrate binding
Binding of substrate & inhibitor is mutually exclusive
Inhibition can be reversed by increasing the concentration of substrate at a constant [I]
Degree of inhibition will depend on the concentrations of substrate & inhibitor and on the relative affinities of the enzyme for substrate & inhibitor Competitive inhibition<br>
slide8. Non-competitive Inhibition An inhibitor that binds to an enzyme to form a dead end complex, whether or not the active site is occupied by a substrate is termed as a Non Competitive Inhibitor
Can bind either to E or ES complex
Since inhibitor doesn't bear structural resemblance to the substrate, it must bind to the enzyme at a site distinct from the substrate binding site
The presence of inhibitor does not affect substrate bonding but does interfere with the catalytic functioning of the enzyme
The binding of inhibitor often deforms the enzyme so that it doesn’t form ES complex at a normal rate and once formed, ES complex doesn’t decompose at normal rate to yield products
Non competitive inhibitor doesn’t affect the Km because the binding of inhibitor does not block substrate binding or vice-versa
Inhibitor effectively lowers the concentration of active enzyme and hence decreases the apparent Vmax
since there is no competition between substrate & inhibitor, the inhibition is not reversed by increasing the [S]<br>
slide9. Lineweaver – Burk Plot<br>
slide10. Uncompetitive Inhibition
Inhibitor doesn't bind to the free Enzyme rather it binds to the ES complex
the binding of an uncompetitive inhibitor is presumed to cause structural distortion of the active site making the enzyme catalytically inactive
the binding of substrate could cause a conformational change in the enzyme thereby revealing an inhibitor binding site
Inhibition can’t be reversed by increasing the [S] since inhibitor doesn't compete with substrate for the same binding site
The equilibria show that at any [I] an infinitely high [S] will not drive all the enzyme to ES form; some non productive ESI complex will always be present. Consequently an uncompetitive inhibitor will decrease the Vmax
An uncompetitive inhibitor will also decrease the Kmapp because the reaction
ES + I ESI removes some ES causing the reaction
E + S ES to proceed to the right<br>
slide12. [I]2 [I]1 No I -1/Kmapp -1/Km Km
Vmax Slope = 1/Vmax 1/Vmaxi 1/v 1/[s]ï‚® Increasing [I] Lineweaver Burk plot 1
v Km 1 1
Vmax [S] Vmax = + (1+ ) [I]
Ki Slope remains Unchanged & Intercept Increases By the factor (1+[ I ] )
Ki Incase of UC Inhibition Ki is that concn of I which halves the value of both Vmax and Km<br>
slide14. Irreversible Inhibition An irreversible inhibitor binds at or near the active site of the enzyme irreversibly, usually by covalent bonds, so that it can’t subsequently dissociate from the enzyme
The inhibitor destroys as essential functional group on the enzyme that participates in normal substrate binding or catalytic action. As a result the enzyme is rendered permanently inactive
Compounds which irreversibly denature the enzyme protein or cause non-specific inactivation of the active site are not usually regarded as irreversible inhibitors.
Irreversible inhibitors are bind via covalent linking to the enzyme causing modification of the enzyme and inactivating it.
Many enzymes contain -SH, -OH,- or -COOH groups as part of their active sites, any chemical which can react with them acts as an irreversible inhibitor. Heavy metals such as Ag+, Hg2+, Pb2+Â have strong affinities for -SH groups.<br>
slide15. Irreversible inhibitors Active site directed irreversible Inhibitors
or
(Affinity labels) Suicide Inhibitors
(Mechanism-based Inhibitors)
or
(kcat Inhibitors) Types of Irreversible Inhibitors<br>
slide16. Affinity labels An affinity label is a chemically reactive compound that is designed to resemble the substrate of an enzyme so that it binds at the active site and forms a stable covalent bond with a susceptible group of the nearby residue in the enzyme protein.
Affinity labels are very useful for identifying catalytically important residues<br>
slide17. Suicide Inhibitors A suicide inhibitor is a relatively inert molecule that is transformed by an enzyme at its active site into a reactive compound that irreversibly inactivates the enzyme
They are substrate analogs designed so that via normal catalytic action of the enzyme, a very reactive group is generated.
The latter forms a covalent bond with a nearby functional group within the active site of the enzyme causing irreversible inhibition.
Such inhibitors are called suicide inhibitors because the enzyme appears to commit suicide and also known as dead end inhibitor .
e.g. FdUMP is a suicide inhibitor of thymidylate synthase.<br>
slide18. For understanding the regulation of enzyme activity within the living cells
To elucidate the kinetic mechanism of an enzyme catalyzing a multisubstrate reaction
Useful in elucidating the cellular metabolic pathways by causing accumulation of intermediates
Identification of the catalytic groups at the active site
Provide information about substrate specificity of the enzyme
Form the basis of drug designing. The whole area of selective toxicity , including the use of antibiotic, toxin, insecticides etc. is based on the exploitation of species differences in the susceptibility to enzyme inhibitors.
Competitive inhibitors are useful in x-rays crystallographic studies to pin point the active site in crystal structure and thus revealing how the surrounding amino acid residues interact with the bound molecule.
To treat methanol poisoning Importance of Enzyme Inhibition<br>
slide2. Enzyme Inhibition/Inhibitor: An Enzyme inhibitor is a compound that decreases or tends to decrease the rate of an enzyme catalyzed reaction by influencing the binding of substrate or its turnover number.
Blocking an enzyme's activity by chemical molecules which resemble or mimic a particular enzymes substrate(s) is called Enzyme Inhibition and the mole is said to be an Inhibitor. Therefore, many therapeutic drugs are some type of enzyme inhibitor.
Molecules that bind to enzymes and increase their activity are called Enzyme Activators. The binding of an inhibitor can stop a substrate from entering the enzyme's active site or hinder the enzyme from catalysing its reaction.
The modes and types of inhibitors have been classified by their kinetic activities and sites of actions.<br>
slide3. Type of Enzyme Inhibitors Reversible Irreversible Type of
Inhibitors Competitive Uncompetitive Non- Competitive Active Site
Directed Suicide
Inhibitors Mixed<br>
slide4. Types of Inhibition 1.Reversible Inhibition:Â
Reversible inhibitors bind non-covalently (hydrogen bonds, hydrophobic interactions and ionic bonds) and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.
Inhibitor binds to Enzyme reversibly through weak non- covelent interactions
An Equilibrium is established between the free inhibitor & EI Complex and is defined by an equilibrium constant (Ki)
The activity of Enzyme is fully restored on removing the inhibitor by dialysis.
Reversible inhibitors depending on concentration of E, S and I, show a definite degree of inhibition which is reached fairly rapidly and remains constant when initial velocity studies are carried out.<br>
slide5. 2.Irreversible Inhibition:Â
Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors modify key amino acid residues needed for enzymatic activity.
Inhibitor binds at or near the active site of the enzyme irreversibly, usually by covalent bonds, so it can’t dissociate from the enzyme
No equilibrium exits
Enzyme activity is not regained on dialysis
Effectiveness of inhibitor is expressed not by equilibrium constant but by a velocity constant, which determines the fraction of the enzyme inhibited in a given period of time by a certain concentration of the inhibitor<br>
slide6. A competitive inhibitor combines with the free enzyme to form an EI complex in a manner that prevents substrate binding
Binding of substrate & inhibitor is mutually exclusive
Inhibition can be reversed by increasing the concentration of substrate at a constant [I]
Degree of inhibition will depend on the concentrations of substrate & inhibitor and on the relative affinities of the enzyme for substrate & inhibitor Competitive inhibition<br>
slide8. Non-competitive Inhibition An inhibitor that binds to an enzyme to form a dead end complex, whether or not the active site is occupied by a substrate is termed as a Non Competitive Inhibitor
Can bind either to E or ES complex
Since inhibitor doesn't bear structural resemblance to the substrate, it must bind to the enzyme at a site distinct from the substrate binding site
The presence of inhibitor does not affect substrate bonding but does interfere with the catalytic functioning of the enzyme
The binding of inhibitor often deforms the enzyme so that it doesn’t form ES complex at a normal rate and once formed, ES complex doesn’t decompose at normal rate to yield products
Non competitive inhibitor doesn’t affect the Km because the binding of inhibitor does not block substrate binding or vice-versa
Inhibitor effectively lowers the concentration of active enzyme and hence decreases the apparent Vmax
since there is no competition between substrate & inhibitor, the inhibition is not reversed by increasing the [S]<br>
slide9. Lineweaver – Burk Plot<br>
slide10. Uncompetitive Inhibition
Inhibitor doesn't bind to the free Enzyme rather it binds to the ES complex
the binding of an uncompetitive inhibitor is presumed to cause structural distortion of the active site making the enzyme catalytically inactive
the binding of substrate could cause a conformational change in the enzyme thereby revealing an inhibitor binding site
Inhibition can’t be reversed by increasing the [S] since inhibitor doesn't compete with substrate for the same binding site
The equilibria show that at any [I] an infinitely high [S] will not drive all the enzyme to ES form; some non productive ESI complex will always be present. Consequently an uncompetitive inhibitor will decrease the Vmax
An uncompetitive inhibitor will also decrease the Kmapp because the reaction
ES + I ESI removes some ES causing the reaction
E + S ES to proceed to the right<br>
slide12. [I]2 [I]1 No I -1/Kmapp -1/Km Km
Vmax Slope = 1/Vmax 1/Vmaxi 1/v 1/[s]ï‚® Increasing [I] Lineweaver Burk plot 1
v Km 1 1
Vmax [S] Vmax = + (1+ ) [I]
Ki Slope remains Unchanged & Intercept Increases By the factor (1+[ I ] )
Ki Incase of UC Inhibition Ki is that concn of I which halves the value of both Vmax and Km<br>
slide14. Irreversible Inhibition An irreversible inhibitor binds at or near the active site of the enzyme irreversibly, usually by covalent bonds, so that it can’t subsequently dissociate from the enzyme
The inhibitor destroys as essential functional group on the enzyme that participates in normal substrate binding or catalytic action. As a result the enzyme is rendered permanently inactive
Compounds which irreversibly denature the enzyme protein or cause non-specific inactivation of the active site are not usually regarded as irreversible inhibitors.
Irreversible inhibitors are bind via covalent linking to the enzyme causing modification of the enzyme and inactivating it.
Many enzymes contain -SH, -OH,- or -COOH groups as part of their active sites, any chemical which can react with them acts as an irreversible inhibitor. Heavy metals such as Ag+, Hg2+, Pb2+Â have strong affinities for -SH groups.<br>
slide15. Irreversible inhibitors Active site directed irreversible Inhibitors
or
(Affinity labels) Suicide Inhibitors
(Mechanism-based Inhibitors)
or
(kcat Inhibitors) Types of Irreversible Inhibitors<br>
slide16. Affinity labels An affinity label is a chemically reactive compound that is designed to resemble the substrate of an enzyme so that it binds at the active site and forms a stable covalent bond with a susceptible group of the nearby residue in the enzyme protein.
Affinity labels are very useful for identifying catalytically important residues<br>
slide17. Suicide Inhibitors A suicide inhibitor is a relatively inert molecule that is transformed by an enzyme at its active site into a reactive compound that irreversibly inactivates the enzyme
They are substrate analogs designed so that via normal catalytic action of the enzyme, a very reactive group is generated.
The latter forms a covalent bond with a nearby functional group within the active site of the enzyme causing irreversible inhibition.
Such inhibitors are called suicide inhibitors because the enzyme appears to commit suicide and also known as dead end inhibitor .
e.g. FdUMP is a suicide inhibitor of thymidylate synthase.<br>
slide18. For understanding the regulation of enzyme activity within the living cells
To elucidate the kinetic mechanism of an enzyme catalyzing a multisubstrate reaction
Useful in elucidating the cellular metabolic pathways by causing accumulation of intermediates
Identification of the catalytic groups at the active site
Provide information about substrate specificity of the enzyme
Form the basis of drug designing. The whole area of selective toxicity , including the use of antibiotic, toxin, insecticides etc. is based on the exploitation of species differences in the susceptibility to enzyme inhibitors.
Competitive inhibitors are useful in x-rays crystallographic studies to pin point the active site in crystal structure and thus revealing how the surrounding amino acid residues interact with the bound molecule.
To treat methanol poisoning Importance of Enzyme Inhibition<br>