Chapter 8: Enzymes: Kinetics - Bulacan State University, College of Medicine

BULACAN STATE UNIVERSITY COLLEGE OF MEDICINE DEPARTMENT OF BIOCHEMISTRY CHAPTER 8: ENZYMES: KINETICS ALVIN G. MARIANO, RN, MD, DPPS  LEARNING OBJECTIVES At the end of the lesson, you should be able to: Understand the Michaelis-Menten Equ...

BULACAN STATE UNIVERSITY COLLEGE OF MEDICINE DEPARTMENT OF BIOCHEMISTRY CHAPTER 8: ENZYMES: KINETICS ALVIN G. MARIANO, RN, MD, DPPS  LEARNING OBJECTIVES At the end of the lesson, you should be able to: Understand the Michaelis-Menten Equation Differentiate between competitive, non-competitive, and uncompetitive inhibition Analyze the significance of enzyme efficiency Explore the role of allosteric regulation in enzyme kinetics  ENZYMES Biologically active proteins Serves as catalyst without being consumed or changed Speeds up a chemical reaction Lowers down activation energy of a reactant HOW DO ENZYMES WORK? Lock and Key Model  BIOMEDICAL IMPORTANCE A balanced set of enzyme activities are important to maintain homeostasis or equilibrium Disturbance in equilibrium results to abnormal rates of reactions / processes leading to diseases Enzymes are targets for drug discovery Enzyme kinetics are principal tools to characterize efficacy of therapeutic agents ENZYME KINETICS The study of the rates of enzyme- catalyzed chemical reactions The reaction rate is measured quantitatively The effect of certain factors on the rate of reaction are evaluated BALANCED CHEMICAL EQUATION A written representation of a chemical reaction Reversible reaction A+B P+Q Irreversible reaction A+B P+Q GIBBS FREE ENERGY CHANGE (△G) The energy associated with a chemical reaction that can be used to work Describes the direction in which the chemical reaction will tend to proceed and the concentration of reactants and products that will be present at equilibrium ACTIVATION ENERGY Minimum energy to start a reaction △GF – change in energy to reach a transition state (transient intermediates) ENERGY CHANGES DURING A REACTION Energy barrier of a reaction Reactant A Transition state Product B High energy intermediate ENERGY CHANGES DURING A REACTION Energy barrier of a reaction Transition state Reactant A Product B ENERGY CHANGES DURING A REACTION Energy of Activation (Eact) △GF to form transition state intermediates Difference between the free energy of the reactant and the transition state ENERGY CHANGES DURING A REACTION RATE OF REACTION For molecule to react, it must have sufficient energy to overcome the energy barrier of the transition state The lower the free energy of activation, the faster the rate of the reaction. ENERGY CHANGES DURING A REACTION ALTERNATE REACTION PATHWAY Enzymes allow a reaction to proceed by lowering the energy of activation Enzyme does not change the free energy of the reactants or products HOW DOES A CHEMICAL REACTION “Kinetic theory or collision theory” of OCCUR? chemical reaction For a chemical reaction to occur, the molecules must: 1. Have sufficient energy 2. Collide in correct orientation or approach each other in bond forming distance EFFECT of TEMPERATURE ON REACTION VELOCITY Temperature Substrate / Reactant concentration Pressure (for gas) Surface area (for solid) Nature of reactants (solid, liquid, or gas) Presence of catalyst  EFFECT of TEMPERATURE ON REACTION VELOCITY Increase temperature Increase in kinetic energy of molecules Rapid motion of molecules Increased frequency of productive As temperature increases collision more molecules have kinetic Increase in rate of reaction energy exceeding the energy barrier.  EFFECT of TEMPERATURE ON REACTION VELOCITY Increase temperature Increase in kinetic energy of molecules Rapid motion of molecules Increased frequency of productive As the temperature increases, collision the rate of the reaction Increase in rate of reaction increases EFFECTS of SUBSTRATE CONCENTRATION on REACTION VELOCITY The higher the number of molecules (substrate), the higher the probability of collision EFFECTS of SUBSTRATE CONCENTRATION on REACTION VELOCITY The rate of reaction is proportional to the concentration of the molecules  FACTORS AFFECTING REACTION VELOCITY Temperature – particles move factors, increases collision Concentration – more particle molecules, more collision Surface area – more particles are exposed to the other reactant Pressure – for gases, increasing the pressure causes more collision of molecules Nature of reactants – reactants of same nature with have faster rate of reaction Catalyst – presence of catalyst reduces energy of activation KINETIC ORDER OF REACTION  PROPERTIES OF A SYSTEM AT EQUILIBRIUM The overall concentrations of reactants and products remain constant The rate of conversion of substrates to products is equal to which the products are converted to substrate Rate1 = Rate-1 The equilibrium constant (Keq) is a ratio of the reaction rate constant of the forward reaction (K1) to that of the reverse reaction (K-1) Keq = K1 K-1  PROPERTIES OF ENZYME KINETICS Enzymes lower the activation energy (△GF) for the formation of transition state intermediates Enzymes undergo transient modifications during the catalytic activity but emerge unchanged by the end of the reaction It has no effect on the free energy of the reactants (△G0) and on the equilibrium constant (KEq) FACTORS AFFECTING ENZYME CATALYZED REACTIONS Temperature Hydrogen ion concentrations (pH) Substrate concentration Presence of inhibitors EFFECT of TEMPERATURE ON ENZYME CATALYZED REACTIONS Increase in temperature increases reaction velocity by increasing kinetic energy and collision frequency Beyond the optimum temperature, reaction slows down, as enzymes are denatured losing its catalytic activity Enzymes in human exhibit stability at temperature up to 45 – 550C  EFFECTS OF TEMPERATURE ON ENZYME CATALYZED REACTIONS TEMPERATURE COEFFICIENT (Q10) The factor by which the rate of biological process increases for a 10 0C increase in temperature The rates of most biological process typically double for a 10 0C rise in temperature (Q10 = 2) Survival feature of cold-blooded animals For mammals, changes in enzyme reactions occur during fever or hypothermia EFFECT OF HYDROGEN ION CONCENTRATION (pH) on ENZYME CATALYZED REACTIONS The rate of enzyme catalyzed reactions depends on hydrogen ion concentration The residues involved must be in the charged state Extremes of pH affects the charged state and can denature the enzymes Ex: Enzymes with amino group (NH3+) which is positively charged is deprotonated in an alkaline pH decreasing its catalytic activity EFFECTS OF SUBSTRATE CONCENTRATION ON ENZYME CATALYZED REACTIONS Velocity of the reaction (V) increases as substrate concentration increases until a maximal Velocity (Vmax) is reached At Vmax there is saturation of enzyme binding sites Hyperbolic curve EFFECT of INHIBITORS ON ENZYME CATALYZED REACTIONS Competitive Inhibition Non-competitive Inhibition  EFFECTS OF ENZYME INHIBITORS Competitive Inhibition Non-competitive Inhibition  MICHAELIS-MENTEN EQUATION MAUDE LEONORA MENTEN and LOENOR MICHAELIS Formulated the Michaelis-Menten equation to describe the steady state action of enzymes  MICHAELIS – MENTEN EQUATION Illustrates the relationship of initial reaction Velocity (Vi) with substrate concentration Michaelis constant (Km) is the substrate concentration at which velocity is ½ Vmax Vi = initial reaction velocity Vmax = maximal velocity Km = Michaelis constant Vi=Vmax (S) (S) = Substrate concentration Km + (S)  MICHAELIS – MENTEN EQUATION EFFECT OF SUBSTRATE CONCENTRATION 1. When (S) is less than Km, the initial reaction velocity is directly proportional to the (S) Vi = initial reaction velocity Vmax = maximal velocity Km = Michaelis constant Vi=Vmax (S) (S) = Substrate concentration Km + (S)  MICHAELIS – MENTEN EQUATION EFFECT OF SUBSTRATE CONCENTRATION 2. When (S) is much greater than Km, the reaction velocity is maximal and unaffected by further increase in the (S) Vi = initial reaction velocity Vmax = maximal velocity Km = Michaelis constant Vi=Vmax (S) (S) = Substrate concentration Km + (S)  MICHAELIS – MENTEN EQUATION EFFECT OF SUBSTRATE CONCENTRATION 3. When (S) is equal to Km, the initial reaction velocity half maximal Vi = initial reaction velocity Vmax = maximal velocity Km = Michaelis constant Vi=Vmax (S) (S) = Substrate concentration Km + (S)  CONCLUSIONS ABOUT MICHAELIS – MENTEN EQUATION 1. Characteristics of Km (Michaelis constant) Km = (S) at ½ Vmax Reflects affinity of the enzyme for that substrate Vi=Vmax (S) Km + (S)  CONCLUSIONS ABOUT MICHAELIS – MENTEN EQUATION 1. Characteristics of Km (Michaelis constant) Small Km = High affinity Small amount of substrate to saturate the enzymes Large Km = Low affinity Larger amount of substrate to saturate the enzymes Vi=Vmax (S) Km + (S)  CONCLUSIONS ABOUT MICHAELIS – MENTEN 2. Relationship of velocity to EQUATION enzyme concentration The rate of reaction is directly proportional to the enzyme concentration If the amount of enzyme is halved, Vi and Vmax is also halved. Vi=Vmax (S) Km + (S)  CONCLUSIONS ABOUT MICHAELIS – MENTEN 3. Order of Reaction EQUATION FIRST ORDER REACTION If S is > Km, the velocity of reaction is constant and is equal to Vmax Vi=Vmax (S) Km + (S)  LINEWEAVER – BURK PLOT A linear form of Michaelis– Menten equation When 1/V is plotted against 1/S, a straight line is obtained (Lineweaver- Burk plot or double reciprocal plot) and it is now possible to calculate Km and Vmax EFFECT of ENZYME INHIBITORS on Km and Vmax Competitive Inhibition Non-competitive Inhibition EFFECT of COMPETITIVE INHIBITORS on Km and Vmax EFFECT OF NON-COMPETITIVE INHIBITORS on Km and VMAX Vmax ½ Vmax EFFECT OF (+) EFFECTORS on Km and VMAX Km  EFFECT ON MICHELIS – MENTEN KINETICS Effect of effectors and inhibitors on Km and Vmax Positive effector EFFECT of INHIBITORS on the LINEWEAVER – BURK COMPETITIVE INHIBITION PLOT A competitive inhibitor moves the Km towards 0, and the line intercepts slope at 1/Vmax Km is increased Vmax is unchanged EFFECT of INHIBITORS on the LINEWEAVER – BURK NON-COMPETITIVE INHIBITION PLOT A non-competitive inhibitor moves Vmax away from 0, and the line intercepts the slope at 1/Km Km is unchanged Vmax is decreased  COMPETITIVE INHIBITION Km is increased Vmax is unchanged  NON - COMPETITIVE INHIBITION Km is unchanged Vmax is decreased  ENZYME KINETICS OF SOME INHIBITORS TIGHTLY BOUND INHIBITORS Binds to enzyme with very high affinity A significant fraction of EI complex exist that it violates the steady state kinetics For kinetic analysis, the kinetic equation should include concentration of the enzyme inhibitor to estimate the reaction constant Ki This enzymes can still be recovered by breaking the EI bond  ENZYME KINETICS OF SOME INHIBITORS IRREVERSIBLE INHIBITORS “POISON ENZYME” Acts irreversibly by chemically modifying the enzyme Involves making or breaking covalent bonds with aminoacyl residues essential for substrate binding and catalytic activity The enzyme is altered permanently and remains inhibited even after removal of the inhibitor EX: heavy metal atom or acylating agent  ENZYME KINETICS OF SOME INHIBITORS MECHANISM BASED or “SUICIDE” INHIBITORS The inhibitor is specialized substrate analog that contains a chemical group that transforms the catalytic machinery of the target enzyme It blocks the function of the catalytic residue Promising lead to the development of enzyme specific drugs  MULTISUBSTRATE ENZYMES Follows Michaelis Menten Equation SEQUENTIAL or SINGLE DISPLACEMENT REACTIONS Both substrates must combine with the enzyme to form a tertiary complex before catalysis can proceed Random or compulsory order In random order, substrate A or B may combine with the enzyme to form EA or EB In compulsory order, substrate A must combine with the enzyme before substrate B can combine with the EA complex  MULTISUBSTRATE ENZYMES Follows Michaelis Menten Equation SEQUENTIAL or SINGLE DISPLACEMENT REACTIONS Random Order  MULTISUBSTRATE ENZYMES Follows Michaelis Menten Equation SEQUENTIAL or SINGLE DISPLACEMENT REACTIONS Compulsory Order Reaction  MULTISUBSTRATE ENZYMES PING-PONG REACTION A mechanism in which one or more products are released from the enzyme before all the substrates have been added Double – displacement reaction Substrate A binds with the enzyme to form Product P, the enzyme is then modified to combine with Substrate B to form product Q, then the enzyme is regenerated  MULTISUBSTRATE ENZYMES PING-PONG REACTION  CLINICAL APPLICATION Enzymes are targets for development of drugs Manu drugs act as enzyme inhibitors 1. Destroys or impairs development, growth, and invasiveness of pathogens 2. Stimulated endogenous defense 3. Impede aberrant molecular processes triggered by genetic, environmental, or biological stimuli with minimal disturbance of the host’s normal cellular function Applying enzyme kinetics, drug effectivity can be determined  SUMMARY Enzymes are protein catalysts which accelerates chemical reaction Enzymes has active and regulatory sites which binds with substrates and inhibitors Substrates has high energy barrier that must be overcome for a reaction to proceed Enzymes lower the free energy of activation of substrate  SUMMARY Most enzymes shows a Michaelis-Menten kinetics, hyperbolic curve Km, the amount of substrate to achieve ½ Vmax Vmax, maximum velocity achieved given a constant amount of enzyme S > Km, the reaction velocity is constant and is equal to Vmax (Zero Order Rx)  SUMMARY If 1/V is plotted against 1/S, a straight line is obtained, the Lineweaver Burk Plot, where 1/Km intersect the X axis and 1/Vmax intersect the Y axis A competitive inhibitor increases Km while Vmax is unchanged A non-competitive inhibitor decreases Vmax while Km is unchanged  SUMMARY In the Lineweaver Burk Plot: A competitive inhibitor moves the Km towards O, and the line intercepts slope at 1/Vmax A non-competitive inhibitor moves Vmax away from 0, and the line intercepts the slope at 1/Km Knowledge on enzyme kinetic can enhance formulation of highly effective drugs THANK YOU!