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MGD module-S2 Session three 2022-2023 Enzymes Activity Dr. Hishyar A. Najeeb, MSc., PhD (UK) Assistant professor Clinical Biochemistry & Molecular Medicine References Ø Ø Ø Marks’ Basic Medical Biochemistry Chapters 8, 9, 45 Medical Biochemistry Chapters 5, 6 Lippincott’s Illustrated Reviews: B...

MGD module-S2 Session three 2022-2023 Enzymes Activity Dr. Hishyar A. Najeeb, MSc., PhD (UK) Assistant professor Clinical Biochemistry & Molecular Medicine References Ø Ø Ø Marks’ Basic Medical Biochemistry Chapters 8, 9, 45 Medical Biochemistry Chapters 5, 6 Lippincott’s Illustrated Reviews: Biochemistry Chapter 5 2 Enzyme Activity & Kinetics Inhibition 3 Lecture 5 Learning outcomes 1) Explain the effects of enzymes on chemical reactions. 2) Describe how reaction rates vary as a function of enzyme and substrate concentration. 3) Define the terms activity, international unit of enzyme activity, Km and Vmax. 4) Analyse and interpret kinetic data for enzymecatalysed reactions. 5) Describe the effects of enzyme inhibitors on enzyme kinetics and be able to distinguish between the two from simple graphs. 4 Effect of Enzyme on Chemical Reaction There are two fundamental conditions for life. First, the living thing must be able to selfreplicate. Second, the organism must be able to catalyze chemical reactions efficiently and selectively. Many of us, for example, consume substantial amounts of sucrose as a kind of fuel. The conversion of sucrose to CO2 and H2O in the presence of oxygen is a highly exergonic process, releasing free energy that we can use to think, move, taste, and see. However, a bag of sugar can remain on the shelf for years without any observable conversion to CO2 and H2O. 5 Enzyme Nomenclature Enzymes are named by the type of reaction that they catalyse. Usually this means adding the suffix –ase to the name of their substrate or reaction that they catalyse e.g. Lactase hydrolyses lactose into glucose and galactose DNA polymerase, polymerises deoxynucleotides to form DNA 6 Six Major Classes of Enzymes 7 8 Properties of enzymes 1. Virtually all enzymes are proteins Some enzymes also require the presence of additional chemical components to catalyse reactions. *Cofactors are inorganic ions such as Fe2+, Mn2+etc. *Coenzymes are organic compounds that act as temporary carriers of groups in the reaction e.g. nicotinamide adenine dinucletide (NAD), Coenzyme A (CoA). *Coenzymes or cofactors that are tightly or covalently linked to the enzyme protein are known as prosthetic groups. 2. Enzymes are highly specific Interact with one or only a few substrates and catalyse one type of reaction. 9 The protein part of the enzyme (apo-enzyme) + Cofactor, prosthetic group, or the coenzyme =Holoenzyme Holoenzyme 10 3. Enzymes increase the rate of a reaction They DO NOT affect the equilibrium of a reaction. 4. Enzymes are left unchanged after the reaction has occurred. 11 How do Enzymes Work? Ø Ø Ø Enzyme-catalyzed reactions are characterized by the formation of a complex between substrate and enzyme (an ES complex). Substrate binding occurs in a pocket on the enzyme called the active site. The function of enzymes and other catalysts is to lower the activation energy for a reaction to occur. 12 How do Enzymes Work? Ø Ø Enzymes work by lowering the activation energy needed for a reaction to occur. Binding of substrate to a distinct part of the enzyme, the active site, increases the local concentration of reactants and also stabilises the formation of the high energy transition state. 13 Reaction Rates as a Function of Enzyme and Substrate Conc Ø The relationship between [S] and Vo has the general shape for most enzymes (hyperbolic), which can be expressed by the Michaelis-Menten equation: WhereV0= initial reaction velocity [S] = substrate concentration Vmax = maximal velocity Km = Michaelis constant *Low Km means high affinity of the enzyme to the substrate. *High Km means low affinity of the enzyme to the substrate. 14 Effect of Substrate Conc. Effect of substrate concentration on the initial velocity of an enzymecatalyzed reaction. 15 Reaction Rates as a Function of Enzyme and Substrate Conc. Lineweaver-Burk Equation Inverting the Equation yields Lineweaver-Burke Equation : 16 Reaction Rates as a Function of Enzyme Conc. Ø Ø The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations. For example, if the enzyme concentration is halved, the initial rate of the reaction as well as that of are reduced to one half that of the original. 17 Factors Affecting Reaction Velocity 1)Substrate concentration Increase in S conc. =Increase in the rate of reaction to a certain point 18 2) Effect of Temp. 19 3) Effect of pH 20 International unit of Enzyme Activity Ø Ø Ø In some diseases, there may be a deficiency or even a total absence of one or more enzymes. For other disease conditions, excessive activity of an enzyme may be the cause. Measurements of the activities of enzymes in blood plasma, erythrocytes, or tissue samples are important in diagnosing certain illnesses By international agreement, 1.0 unit of enzyme activity is defined as the amount of enzyme causing transformation of 1.0 micromol of substrate per minute, under optimal conditions of measurement. The term activity refers to the total units of enzyme in a solution. 21 Inhibition of enzyme activity Many drugs work by inhibiting the activity of enzymes. 1-Irreversible inhibitors: Bind covalently to the enzyme molecule to destroy enzyme function 22 2-Reversible inhibitors i) Competitive inhibitors: -Binds at the active site -Affects Km not Vmax -Can be overcome by increasing the substrate concentration 23 Examples of competitive inhibitors: Ø Ø Allopurinol competes with hypoxanthine for xanthine oxidase inhibiting the formation of uric acid, so it is used in treatment of hyperuricemia (gout). Statins (e.g. atorvastatin) competes with HMGCoA for its reductase, so, it inhibits cholesterol synthesis. 24 ii) Non-competitive inhibitors Binds at a site other than the active site -Affects Vmax not Km -Cannot be overcome by increasing the substrate concentration. 25 26