1. ENZYME INHIBITION Studies on Inhibitors are useful for:Studies on Inhibitors are useful for: Mechanistic studies to learn about how enzymes interact with their substrates.Mechanistic studies to learn about how enzymes interact with their substrates. Role of inhibitors in enzyme regulation.Role of inhibitors in enzyme regulation. Drugs if they inhibit abberrant biochemical reactions:Drugs if they inhibit abberrant biochemical reactions: –penicillin, ampicillin, et al.: interfere with the synthesis of bacterial cell walls. –methotrexate: anti-cancer drug that affects DNA metabolism in actively growing cells Understanding the role of biological toxins.Understanding the role of biological toxins. –Arsenate: mimics phosphate esters in enzyme reactions, but are easily hydrolysed. –Amino acid analogs: useful herbicides (i.e. roundup) –Insecticides: chemicals targeted for the insect nervous system.

2. TYPES OF INHIBITION REVERSIBLE INHIBITIONREVERSIBLE INHIBITION They are of three types 1. Competitive Inhibition 2. Non Competitive Inhibition and 3. Un Competitive Inhibition IRREVERSIBLE INHIBITIONIRREVERSIBLE INHIBITION ALLOSTERIC INHIBITIONALLOSTERIC INHIBITION

3. 1.COMPETITIVE INHIBITION Inhibitor binds to the same site on the enzyme as the substrate, the active site.Inhibitor binds to the same site on the enzyme as the substrate, the active site. Inhibitor only binds to the free enzyme.Inhibitor only binds to the free enzyme. Inhibitor is usually structurally very similar to the substrate. Examples:Inhibitor is usually structurally very similar to the substrate. Examples: –Succinate / Malonate –ATP/AMP The reaction scheme that corresponds to competitive inhibition is:The reaction scheme that corresponds to competitive inhibition is:

4. The inhibitor reduces the amount of E available for productive catalysis by the formation of the EI complex. The inhibitor does not affect the ES complex after it has formed. The dissociation constant for the inhibitor is KI = [E][I]/[EI].The inhibitor reduces the amount of E available for productive catalysis by the formation of the EI complex. The inhibitor does not affect the ES complex after it has formed. The dissociation constant for the inhibitor is KI = [E][I]/[EI]. There are two anticipated consequences of this additional competitive equilibrium:There are two anticipated consequences of this additional competitive equilibrium: Vmax is unchanged: At high levels of substrate all of the inhibitor is displaced by substrate.Vmax is unchanged: At high levels of substrate all of the inhibitor is displaced by substrate. KM is increased: Higher substrate concentrations are required to reach the maximal velocity.KM is increased: Higher substrate concentrations are required to reach the maximal velocity.

7. 2.NON COMPETITIVE INHIBITION Noncompetitive Inhibition:Noncompetitive Inhibition: In this case the inhibitor binds to both E and ES. Both the slope (KM/Vmax), and the Y-intercept (1/Vmax) of the Lineweaver-Burk plot increase (see figure 5.11). The KI ('s) are determined as above by replotting the slope and intercept values vs. [I].In this case the inhibitor binds to both E and ES. Both the slope (KM/Vmax), and the Y-intercept (1/Vmax) of the Lineweaver-Burk plot increase (see figure 5.11). The KI ('s) are determined as above by replotting the slope and intercept values vs. [I]. Vmax is decreased: At high levels of substrate the inhibitor is still bound.Vmax is decreased: At high levels of substrate the inhibitor is still bound. KM is increased: Higher [S] is required to reach the lower maximal velocity.KM is increased: Higher [S] is required to reach the lower maximal velocity.

10. 3.UN COMPETITIVE INHIBITION The inhibitor binds directly to the ES complex.The inhibitor binds directly to the ES complex. The inhibitor does not have to bind at the active site.The inhibitor does not have to bind at the active site. The inhibitor does not have to resemble the substrate (e.g. an allosteric inhibitor).The inhibitor does not have to resemble the substrate (e.g. an allosteric inhibitor). Vmax is reducedVmax is reduced The slopes of the Lineweaver-Burk plot (KM/Vmax) are unchanged, but the Y-intercept increases by a factor of (1 + [I]/KI). The X-intercept shifts to the left by a factor (1 + [I]/KI).The slopes of the Lineweaver-Burk plot (KM/Vmax) are unchanged, but the Y-intercept increases by a factor of (1 + [I]/KI). The X-intercept shifts to the left by a factor (1 + [I]/KI).

11. ALLOSTERIC INHIBITION This type of inhibition is exclusive for allosteric enzymes.This type of inhibition is exclusive for allosteric enzymes. Allosteric enzymes differs in their structure from other enzymes by possesing another site apart from active site. This site is called allosteric site where modulators can bindAllosteric enzymes differs in their structure from other enzymes by possesing another site apart from active site. This site is called allosteric site where modulators can bind Modulators may be of two types,Modulators may be of two types, Positive Modulator andPositive Modulator and Negative ModulatorNegative Modulator

12. Contd.. The rate determining pathways in most of the metabolic reactions are catalysed by Allosteric enzymesThe rate determining pathways in most of the metabolic reactions are catalysed by Allosteric enzymes The significance of this specialty is to regulate the metabolic pathway.The significance of this specialty is to regulate the metabolic pathway. Allosteric enzymes do not follow MM-KineticsAllosteric enzymes do not follow MM-Kinetics