SCH2232 Medical Biochemistry Enzyme Kinetics PDF

School of Medical & Health Sciences Chapter 4, 6 SCH2232 Medical Biochemistry Enzyme kinetics Andrew J. Woo, PhD. Today’s lecture: Enzyme Enzymes Enzymes: Reaction Thermodynamics Enzyme Kinetics Enzyme Inhibition  Enzymes What are enzymes?...

School of Medical & Health Sciences Chapter 4, 6 SCH2232 Medical Biochemistry Enzyme kinetics Andrew J. Woo, PhD. Today’s lecture: Enzyme Enzymes Enzymes: Reaction Thermodynamics Enzyme Kinetics Enzyme Inhibition  Enzymes What are enzymes? S+E [ES] P+E Enzymes are catalysts. Significantly increase reaction rates without being used up Most enzymes are globular proteins. However, some RNA (ribozymes and ribosomal RNA) also catalyze reactions. The study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s. The study of enzymes has dominated biochemistry in the past and continues to do so.  Enzymes enzymes in human body moslty - pH 7.4+/- 0.05, T= 37 +/-0.5C (doesn't favour alkalosis or acidosis) Why Biocatalysis over Inorganic Catalysts? Desired & Greater reaction specificity: avoids side Favorable. products Enzyme Milder reaction conditions: conducive to conditions in cells pH ~ 7, 37°C Higher reaction rates: in a biologically useful timeframe Capacity for regulation: control of biological pathways Many potential pathways of decomposition.  Enzymes How do enzymes work? Common cellular processes are slow or unfavourable if uncatalysed Enzyme catalysed reactions occur in active site Molecule bound to enzyme and acted on = substrate All chemical reactions proceeds through one or more high energy transition states Enzymes and other catalysts accelerate the rate of a reaction (105 to 1017 times increase) by reducing the free energy of the transition state therefore lowering activation energy  Enzymes Chemical reactions can go forward and backward  Enzymes page 104 Chemical equilibrium versus steady-state Equilibrium Individual chemical reactions carried out in a test tube will eventually reach an equilibrium Steady State The rate of formation of a favours one direction product is equal to the rate of its consumption, the concentration of the substance remains constant in all linked reactions In Cells, individual reactions may not reach equilibrium Steady state reaction prevents accumulation of excess intermediates.  Enzymes: Reaction Thermodynamics Free energy 1st law of thermodynamics – energy is neither created nor destroyed. G = change is free energy G = negative = energy released Exergonic reaction Enzyme G = positive = energy required Endergonic reaction G = 0 Reaction is in Equilibrium released = absorbed  Enzymes: Reaction Thermodynamics Plotting free energy against reaction progress Reaction coordinate diagram Y-axis = Free energy X-axis = Progressive chemical changes Substrate (S) Enzyme Transition state (‡) – eg bond breakage/formation molecular moment where decay to substrate or decay to product are equally likely Product (P) G’o = overall standard free-energy change in the direction S  P.  Enzymes: Reaction Thermodynamics Plotting free energy against reaction progress Reaction coordinate diagram Y-axis = Free energy X-axis = Progressive chemical changes Substrate (S) Transition state (‡) – eg bond complex breakage/formation Product (P) G’o = overall standard free-energy change in the direction S  P. ∆G‡cat = Activation energy for is lower when Enzyme catalyzes the reaction  Enzymes: Reaction Thermodynamics Enzymes bind transition states best. The idea was proposed by Linus Pauling in 1946. Enzyme active sites are complimentary to the transition state of the reaction. Enzymes bind transition states better than substrates. Stronger/additional interactions with the transition state as compared with the ground state lower the activation barrier. Most important approach to understanding enzyme mechanisms is to determine the rate of a reaction and how it changes in response to changes in experimental parameters Enzymes Kinetics Today’s lecture: Enzyme Enzymes Enzymes: Reaction Thermodynamics Enzyme Kinetics Enzyme Inhibition  Enzyme Kinetics Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes Purpose of studying enzyme kinetics is.. Quantitative description of biocatalysis * zero order 1st order- y=mx second order- y= mx2 Determine the order of binding of substrates Elucidate acid-base catalysis Understand catalytic mechanism Find effective inhibitors drug discovery Understand regulation of activity disease Enzyme Kinetics Working out the Reaction velocity – the speed of reaction Steady state assumption Assume total enzyme concentration is constant Assume total substrate concentration is constant → → Enzyme + Substrate  ES complex  Enzyme + Product → → Products rarely go back to E+S  ES E+P reactants because these are thermodynamically stable ie, Reversal is negligible Through identifying constraints and assumptions, and carrying out long algebra.. We can work out the speed of reaction or Reaction velocity (v) Enzyme Kinetics Working out the Reaction velocity – the speed of reaction Steady state assumption Assume total enzyme concentration is constant Assume total substrate concentration is constant → → Enzyme + Substrate  ES complex Enzyme + Product → → E+S  ES E+P Through identifying constraints and assumptions, and carrying out long algebra.. We can work out the speed of reaction or Reaction velocity (v)  Enzyme Kinetics Michaelis-Menten equation k cat [E tot ][S] V max[S] v= = K m + [S] Km + [S] kcat (turnover number): how many substrate molecules one enzyme molecule can convert per second how many substrate molecules one enzyme molecule can convert per second Km (Michaelis constant): an approximate measure of a substrate’s affinity for an enzyme During steady state, the maximum velocity (Vmax) occurs when all of the enzyme is in the ES complex and is dependent on the breakdown of that complex (k[ES]).  Enzyme Kinetics Michaelis-Menten equation k cat [E tot ][S] V max[S] v= = K m + [S] Km + [S] enzyme saturared Km is substrate concentration [S] where the reaction velocity (V0) is ½ of Vmax = Km Km = ½ Vmax -csubstrate conc  Enzyme Kinetics Effects of substrate concentration on reaction velocity Vmax [ S ] v= Km + S At low substrate concentration [S], reaction velocity (V0) exhibits a linear dependence on [S] At high substrate concentration [S], reaction velocity (V0) becomes maximum reaction velocity (Vmax)  Enzyme Kinetics Effects of substrate affinity on reaction velocity Vmax [ S ] v= Km + S Km = ½ Vmax For high affinity substrate S Km ([S] where ½Vmax ) is lower Low affinity substrate S’ Km is higher (require more S) Vmax eventually is the same.  Enzyme Kinetics Michaelis-Menten equation – Important concept Vmax [ S ] v= Km + S Km = ½ Vmax Km = Michaelis Menten constant = [S] at ½ Vmax The lower the value of Km, the tighter the substrate binding Km can be a measure of the affinity of Enzyme for Substrate  Enzyme Kinetics Active site of an enzyme consists of two functionally important regions: Substrate binding site that Step 1: recognises and binds the → E+S  ES substrate (or substrates) and Fast and reversible Catalytic site whose catalytic Step 2: → ES  E+P groups mediate the chemical Slower, rate limiting step reaction on the substrate Therefore overall rate is proportional to [ES]  Enzyme Kinetics page 550 Measurement of Km And Vmax  Enzyme Kinetics Measurement of Km And Vmax The double-reciprocal Lineweaver-Burk plot is a linear transformation of the Michaelis- Menten plot (Plot of 1/Vo versus 1/[S]) Slope is Km/Vmax Y-intercept is 1/Vmax Easier to work out fundamentals of reaction (including Vmax) using this plot  Enzyme Kinetics Two-Substrate Reactions: Sequential Kinetic Mechanism Kinetic mechanism: the order of binding of substrates and release of products When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms: – sequential mechanism – ping-pong mechanism  Enzyme Kinetics Two-Substrate Reactions: Sequential Kinetic Mechanism Enzyme reaction involving a ternary complex We cannot easily distinguish random from ordered. Random mechanisms in equilibrium will give the Intersecting lines indicate that a intersection point at the y-axis. formation of ternary complex.  Enzyme Kinetics Two-Substrate Reactions: Ping-Pong Kinetic Mechanism Enzyme reaction in which no ternary complex is formed different Km and Vmax Parallel lines indicate a Ping-Pong (double-displacement) pathway  Enzyme Kinetics pH dependence of enzyme activity: pancreatic alpha amylase 7.5, amylase- = 7 Enzyme to adopt the correct conformation and ensure correct charge is present in catalytic active site. Availability of H, and temperature can have dramatic impacts on enzyme function Example: cell destruction Lysosomal enzyme = works at pH 4.5 Chymotrypsin (pancreatic serine proteases) works at pH 8. Today’s lecture: Enzyme Enzymes Enzymes: Reaction Thermodynamics Enzyme Kinetics Enzyme Inhibition  Enzyme Inhibition Inhibitors are compounds that decrease an enzyme’s activity. Irreversible inhibitors (inactivators) react with the enzyme. One inhibitor molecule can permanently shut off one enzyme molecule. They are often powerful toxins but also may be used as drugs. eg: heavy metals Reversible inhibitors bind to and can dissociate from the enzyme. They are often structural analogs of substrates or products. They are often used as drugs to slow down a specific enzyme. Reversible inhibitor can bind to: the free enzyme and prevent the binding of the substrate. the enzyme-substrate complex and prevent the reaction.  Enzyme Inhibition Competitive Inhibition – lines intersect at the y-axis Competes with substrate for binding – binds active site – does not affect catalysis No change in Vmax; lower affinity apparent increase in KM  Enzyme Inhibition Competitive Inhibition – lines intersect at the y-axis Example: Liver enzyme alcohol dehydrogenase converts methanol → formaldehyde Slow IV infusion of ethanol → methanol competed out lower affinity  Enzyme Inhibition Uncompetitive Inhibition Only binds to ES complex – lines are parallel - does not affect substrate binding, - inhibits catalytic function Slower Rate Decrease in Vmax; Apparent decrease in KM higher affinity No change in KM/Vmax  Enzyme Inhibition Mixed Inhibition – lines intersect left from the y-axis. Binds enzyme with or without substrate ― binds to regulatory site ― inhibits both substrate binding and catalysis lipinkottt biochemistry Mixed inhibitors bind at a separate site, but may bind to either E or ES.  Enzyme Inhibition Mixed Inhibition – lines intersect left from the y-axis. Binds enzyme with or without substrate ― binds to regulatory site ― inhibits both substrate binding and catalysis Decrease in Vmax; apparent change in KM Note: Noncompetitive inhibitors are mixed inhibitors such that there is no change in KM.  Enzyme Inhibition Competitive Uncompetitive Mixed Inhibition Recognise the graph pattern and corresponding type of inhibition