Enzyme Inhibition Lecture Notes PDF

Faculty of Medicine Academic Year: 2024-2025 Year: 1 Semester: 1 Module: Human Body Function (HBF) 102 Enzymes II: Enzyme inhibition & regulation By: Shahenda Mahgoub Associate Professor Department: Biochemistry & Molecular Biology 11/29/2024...

Faculty of Medicine Academic Year: 2024-2025 Year: 1 Semester: 1 Module: Human Body Function (HBF) 102 Enzymes II: Enzyme inhibition & regulation By: Shahenda Mahgoub Associate Professor Department: Biochemistry & Molecular Biology 11/29/2024 22  Objectives 1. Compare between different types of enzyme inhibition (competitive & non-competitive). 2. Explain different methods of enzyme regulation. 11/29/2024 HBF - 102 33  Introduction Enzyme inhibition Any substance that can diminish the velocity of an enzyme-catalysed reaction is called an inhibitor. Inhibitors Irreversible Reversible inhibitors inhibitors 11/29/2024 HBF - 102 44  Introduction ▪ Irreversible inhibitors bind to enzymes through covalent bonds. ▪ Reversible inhibitors typically bind to enzymes through noncovalent bonds. ▪ Thus, dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor, and recovery of enzyme activity. HBF - 102 55  Reversible inhibition The two most common types of reversible inhibition are: Competitive Non-competitive. 11/29/2024 HBF - 102 6  Competitive inhibition ▪ In this type of inhibition, the inhibitor competes with the substrate for the active site. ✓ This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for that site.  Competitive inhibition Formation of E.S complex is reduced while a new E.I complex is formed. Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor. Effect on Km: A competitive inhibitor increases the apparent Km for a given substrate. In the presence of a competitive inhibitor, more substrate is needed to achieve ½Vmax. Effect on the Lineweaver-Burk plot Competitive inhibition shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax (Vmax is unchanged). 11/29/2024 HBF - 102 10 Effect on the Lineweaver-Burk plot The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because -1/Km moves closer to zero from a negative value. HBF - 102 11 Helwan Special Medical Program 12 Clinically useful competitive inhibition  Non-competitive Inhibition Inhibitor does not compete with the substrate for the active site of enzyme instead it binds to another site other than the active site. This type of inhibition is recognized by its characteristic effect on Vmax  Non-competitive Inhibition ▪ Non-competitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. ▪ The non-competitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the reaction from occurring.  Effect on Vmax: Non-competitive inhibition cannot be overcomed by increasing the concentration of substrate. Thus, non competitive inhibitors decrease the apparent Vmax of the reaction. Effect on Km: Non-competitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the non-competitive inhibitor. Effect on Lineweaver-Burk plot Non-competitive inhibition is readily differentiated from competitive inhibition by plotting 1/Vo versus 1/[S] and noting that: 1. The apparent Vmax decreases in the presence of a non-competitive inhibitor 2. whereas Km is unchanged. 11/29/2024 HBF - 102 17 Helwan Special Medical Program 18 HBF - 102  Examples on Non-competitive Inhibition 1. The binding of the heavy metal shows non-competitive inhibition. ▪ Ferrochelatase, an enzyme that catalyses the insertion of Fe+2 into protoporphyrin (a precursor of heme, is an example of an enzyme sensitive to inhibition by lead.  Examples on Non-competitive Inhibition 2. Allosteric Inhibition of hexokinase by glucose 6 phosphate ▪ It binds to a different site on the enzyme, changing the shape of the enzyme so that it cannot bind to the substrate effectively. ▪ This slows down the reaction by making the enzyme less active.  Regulation Of Enzyme Activity A.Regulation of allosteric enzymes ▪ Allosteric enzymes are regulated by molecules called effectors (modifiers) that bind noncovalently at a site other than the active site. ▪ The presence of an allosteric effector can alter the affinity of the enzyme for its substrate, or modify the maximal catalytic activity of the enzyme, or both. HBF - 102 23  A-Regulation of allosteric enzymes ▪ Effectors that inhibit enzyme activity are termed negative effectors. ▪ Effectors that increase enzyme activity are called positive effectors. HBF - 102 24 A.Regulation of allosteric enzymes 1-Homotropic effectors: ▪ When the substrate itself serves as an effector, the effect is homotropic. ▪ Most often, an allosteric substrate functions as a positive effector. HBF - 102 25 A.Regulation of allosteric enzymes 2-Heterotropic effectors: The effector may be different from the substrate. e.g., The feedback inhibition. ✓ Feedback inhibition provides the cell with suitable amounts of a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. HBF - 102 26 2-Heterotropic effectors: ▪ The enzyme that converts D to E has an allosteric site that Binds the end-product (G). ▪ If the concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the irreversible step unique to the pathway is inhibited. HBF - 102 27 B-Regulation of enzymes by covalent modification ▪ Many enzymes may be regulated by covalent modification, by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. 11/29/2024 HBF - 102 28 B-Regulation of enzymes by covalent modification Phosphorylation and dephosphorylation: ▪ Phosphorylation reactions are catalysed by a family of enzymes called protein kinases that use adenosine triphosphate (ATP) as a phosphate donor. 11/29/2024 HBF - 102 29 B-Regulation of enzymes by covalent modification ▪ Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases. ▪ Depending on the enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. 11/29/2024 HBF - 102 30 B-Regulation of enzymes by covalent modification ▪ For example, phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases its activity. 11/29/2024 HBF - 102 31 B-Regulation of enzymes by covalent modification ▪ while the addition of phosphate to glycogen synthase (an enzyme that synthesizes glycogen) decreases its activity. 11/29/2024 HBF - 102 32 C-Induction and repression of enzyme synthesis ▪ The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total active sites. ▪ Enzymes subjected to regulation of synthesis are those needed under selected physiologic conditions. 11/29/2024 HBF - 102 33 C-Induction and repression of enzyme synthesis ▪ For example, elevated levels of insulin because of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism. 11/29/2024 HBF - 102 34 C-Induction and repression of enzyme synthesis Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. 11/29/2024 HBF - 102 35  Interactive Question Captopril inhibitory effect can be relieved by raising the ……………… A.pH B.Substrate concentration C.Inhibitor concentration D.Temperature 11/29/2024 HBF - 102 36 36  Interactive Question Regarding heavy metals as enzyme inhibitors,……… A.They bear structural resemblance to substrate. B.They bind to the active site. C.Km and Vmax are increased. D.Km and Vmax are decreased. E.They bind to different site than active site. HBF - 102 37 37  Summary ✓ The two most commonly encountered types of reversible inhibition are competitive (which increases the apparent Km) and noncompetitive (which decreases the apparent Vmax). ✓ The multi subunit allosteric enzymes typically catalyse the rate- limiting of a pathway. ✓ Allosteric enzymes are regulated by molecules called effectors (also modifiers) that bind noncovalently at a site other than the active site. 11/29/2024 HBF - 102 38 38  Summary ✓ Effectors can be either positive (accelerate the enzyme-catalyzed reaction) or negative (slow down the reaction). ✓ An allosteric effector can alter the affinity of the enzyme for its substrate, or modify the maximal catalytic activity of the enzyme, or both. ✓ Enzymes can also be regulated by covalent modification, and by changes in the rate of synthesis or degradation. 11/29/2024 HBF - 102 39 39  References Ferrier, D. R. (2014). Lippincott’s illustrated reviews. USA: Lippincott Williams & Wilkins. 11/29/2024 HBF - 102 40 40