Enzyme Kinetics (Curtin University) PDF

8. Enzyme Kinetics ANDREW CROWE Faculty of Health Sciences | Curtin Medical School (Thanks to STEVE BOTTOMLEY for some material) CRICOS Provider Code 00301J Learning Objectives When you complete this lecture, and any necessary associated reading, you should be able to: Describe how enzyme velocity m...

8. Enzyme Kinetics ANDREW CROWE Faculty of Health Sciences | Curtin Medical School (Thanks to STEVE BOTTOMLEY for some material) CRICOS Provider Code 00301J Learning Objectives When you complete this lecture, and any necessary associated reading, you should be able to: Describe how enzyme velocity may be measured and importance of initial velocity. Describe the Michaelis-Menten equation & its inherent kinetic parameters Km and Vmax Describe the usefulness of kinetic parameters Describe the molecular basis of some typical enzyme inhibitors and understand the practical implications Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J What is Enzyme Kinetics and why is it useful? Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J What is Enzyme Kinetics? Enzyme kinetics looks at how enzymes affect the rate of chemical reactions The ‘activity’ of an enzyme is the rate, or velocity, of the reaction it catalyses Enzyme ‘activity’, ‘velocity’, and ‘rate’ are all terms that refer to how fast the enzyme can catalyse a reaction Enzymes are measured, and characterised, by their effect on the rate of a reaction In living organisms (in vivo) In biotechnology (in vitro) Enzyme kinetics can assist our understanding of factors/toxins/drugs that affect an enzyme’s activity Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Significance of Enzyme Kinetics Enzyme kinetics can give us information on: Function Purpose Mechanism How regulated Identity of residues in the active site The physiological direction of a reaction in a living cell (and hence, people) based on affinity to different concentrations of substrates Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Significance of Enzyme Kinetics (cont) How an enzyme responds to changes in conditions Determine kinetic constants Km, Vmax, Kcat Characterises isoenzymes – Identification (clinical significance) Can inform about the rates of entire metabolic pathways in the cell Understand how inhibitors (or stimulators) affect enzymes Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Practical Application of kinetics In vitro in clinical laboratories E.g. creatine kinase to monitor heart biotechnology E.g. assay ‘kits’, functional kits, quality control, enzyme modification and design Appearance of creatine kinase (CK) & cardiac tropinin after a myocardial infarction Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Michaelis-Menten Enzyme Kinetics Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Measuring Enzyme Activity Why initial rate? Simple linear maths Avoids complex maths % Reverse reaction in system Partial product inhibition as concentrations change in a fixed system Influenced by many factors including: [S], pH, temperature, activators & inhibitors, [E] (from previous lecture) More reproducible for interpreting steady-state kinetics and reaction mechanisms Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J E + S Steady State ES E + P [P] Concentration Vinit [S] Equilibrium is where rate of forward reaction is equal to rate of reverse reaction Steady state is where rate of change of an intermediate (i.e. ES) is zero (unchanged) [E]t [ES] Steady state ES almost constant [E] Pre state time CRICOS Provider Code 00301J Enzyme Activity & Substrate concentration Measure initial rate vo at each substrate concentration Plot the initial velocity as a function of the substrate concentration (Michaelis Menten Plot) Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Graphical interpretation of kinetics Graphical plots; Determine enzyme kinetic parameters Characterise enzymes Four common types of graphs to plot and determine enzyme kinetic data Michaelis-Menten Plot Lineweaver-Burk 1/vo verses 1/[S] Hanes Plot Initial velocity (vo) verses [S] All have their own advantages & disadvantages [S]/vo verses [S] Eadie-Hofstee Plot vo verses vo/[S] Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J The Michaelis Menten Plot (Initial velocity plotted as a function of the substrate concentration) Faculty of Health Sciences | Curtin Medical School nces CRICOS Provider Code 00301J Michaelis Menten Equation The hyperbolic plot is described by the Michaelis-Menten Equation Vo = Vmax*[S] Km + [S] Vo = initial velocity Km = Michaelis Constant different for each enzyme and substrate Affected by various conditions pH, temp, etc [S] = substrate concentration Vmax = maximum velocity of enzyme Different for each enzyme and substrate Also called limiting velocity Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Michaelis Menten equation Vo = Vmax*[S] Km + [S] What is vo (in terms of %Vmax) when: [S] = Km [S] = 10Km [S]=100Km [S]=1000Km? (See next slide) Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Michaelis Menten equation Vo = Vmax*[S] Km + [S] Example of calculation when [S] = 10 fold higher than Km vo = vo = Faculty of Health Sciences | Curtin Medical School Vmax*10Km Km + 10Km 10Km*Vmax = 11Km 10Vmax = 91%Vmax 11 CRICOS Provider Code 00301J Michaelis Menten Vmax Michaelis-Menten equation Velocity Assume Vmax = k3[ES] V = Vmax [S] (Km + [S]) Km [S] CRICOS Provider Code 00301J Figure 8.5 Velocity C B A [S] CRICOS Provider Code 00301J Figure 8.5 Velocity C B A Km [S] CRICOS Provider Code 00301J Figure 8.5 Velocity C B A [S] CRICOS Provider Code 00301J What happens when enzyme conc reduced? Velocity C [E] ê B A Km [S] CRICOS Provider Code 00301J P 76 Lineweaver-Burk plot [S] could be very high before Vmax reached Take the reciprocal of v and [S] Can estimate Vmax from y intercept Can estimate Km from x intercept Usually more variability in the Low [S] data (Increased pipetting/experimental errors) y = ax + b 1/v 1/Vmax bunching of higher [S] tested. Inversing data creates distorted spread of data points 1/km 1/[S] CRICOS Provider Code 00301J Enzyme Catalysis k1 k2 E + S ⇌ ES ⇀ E + P k-1 This reaction mechanism is used to explain the Michaelis Menten equation and plot. It is valid if: Initial velocity is measured The ES exists A steady state is assumed for [ES] Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Enzyme Kinetic Parameters and how they are useful Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Kinetic Parameters: significance of Km? Gives idea of concentration of substrate required to achieve Vmax and saturate active site of the enzyme Useful for enzyme assays Estimate of affinity of enzyme for substrate Approximate value of intracellular level of substrate Adjust enzyme assay conditions E.g. when measuring amount of enzyme where [S]>>Km Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Kinetic Parameters: Why determine Km? Used to compare enzymes (Isozymes) Indicates the relative suitability of alternate substrates Substrate with lowest Km likely to be preferred substrate Indicates relative efficiency and capacity of enzyme Efficiency = how much is produced in a certain time Capacity = how much (substrate) can be handled by enzyme Indicates alterations of enzyme activity due to inhibitors Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Comparison of Km Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Hexokinase Km Hexokinase operating at maximum velocity & insensitive to [glucose] Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Different Km Activity Normal blood levels cyto aldehyde dehydrogenase Glucokinase Mito aldehyde dehydrogenase Hexokinase [ EtOH] or [Glucose] CRICOS Provider Code 00301J Km for each substrate in the reaction Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Useful Kinetic Parameters Vmax maximum or limiting velocity Estimate Km & calculate Kcat Kcat turnover number Measure of the maximal catalytic activity of the enzyme Amount of substrate converted to product per amount of enzyme in unit time when the enzyme is operating near Vmax kcat = Vmax/[Et] where [Et] = total [Enzyme] Also referred to as the molecular activity of the enzyme Not very useful at low [S] Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Useful kinetic parameters Kcat/Km = specificity constant Estimate the specificity of an enzyme for its substrate(s) Efficiency of enzyme with respect to the diffusion limit Can be used to determine the preferred substrate of an enzyme The closer the value to the diffusion limit the more efficient the enzyme Enzyme activity is generally assumed not to be able to exceed the rate of diffusion of about 1010 M-1 s -1 Useful for comparing the efficiency of different enzymes Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J What can kinetic parameters tell us? Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Inhibition of Enzymes and how it is useful Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Enzyme Inhibition Use of inhibitors helps to understand how enzymes work The active site of an enzyme can be mapped using inhibitors that are structural analogs of substrate Once the active site has been mapped, designing other inhibitors/activators of therapeutic value more efficient Treatment of poisoning may be addressed by less toxic inhibitors Enzymes in metabolic pathways can be inhibited by their immediate products or by products downstream in the pathway, or by products of other pathways This leads to enzyme regulation – usually a reduction rather than on/off Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Enzyme Inhibition At least half of the ten most commonly dispensed drugs act as enzyme inhibitors ACE inhibitors block the enzyme that cleaves angiotensin I to form potent vasoconstrictor angiotensin II; causes vasodilation and reduction in blood pressure B-lactam antibiotics such as penicillin & amoxicillin act by inhibiting enzymes in bacterial cell wall synthesis Statin drugs inhibit key enzyme involved in cholesterol synthesis and thereby lower plasma cholesterol Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Enzyme Inhibition Reversible Competitive Inhibitor binds at same site as substrate When substrate not bound Affects only Km Mixed Inhibitor binds at a site different from substrate With or without substrate bound. Affects Vmax or Km or both Changes Vmax/Km Non-competitive inhibition is a ‘special’ case of mixed inhibition where Vmax is affected but not Km Relatively rare. (beyond scope of this unit as mixed inhibitor) Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Enzyme Inhibition Reversible Uncompetitive Inhibitor binds to ES complex also known as catalytic inhibition affects both Vmax and Km no change on Vmax/Km Irreversible Partial or complete – usually Non-competitive Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Competitive Inhibitors Example: Statin drugs competitively inhibit the first committed step in cholesterol synthesis pathway which is catalysed by HMG-CoA reductase. The statin drugs are structural analogs of natural substrate for this enzyme. Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Competitive Effect is to increase the km of enzyme for its substrate. Michaelis Menten Vmax remains the same Inhibitor Lineweaver Burk 1/Vmax Km Km 1/km CRICOS Provider Code 00301J Competitive Inhibition - example Intervention for methanol or antifreeze poisoning Methanol and ethylene glycol (major component of antifreeze) poisoning are fairly common. The metabolic products of these alcohols are toxic. Alcoholics seeking cheap substitutes Accidental consumption by children and animals Enzyme kinetics shows that methanol and ethylene glycol are substrates of alcohol dehydrogenase Ethanol is less harmful and also is a preferred substrate of this enzyme Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Enzyme Inhibition – remedy An answer is to administer ethanol to a blood concentration of about 0.1% (5 shots in 1 hour). Ethanol effectively works as competitive inhibitor, displacing methanol or ethylene glycol (radiator antifreeze) from the enzyme and allowing time for them to be excreted unchanged through kidneys, thus avoiding toxic effects of the metabolic products of these poisons. Source of information is Stryer’s “Biochemistry and http:jeffline.tju.edu/CWIS/DEPT/biochemistry/ Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Non-competitive (similar to reduction of enzyme conc) Vmax C [E] ê B A 1/Vmax Km 1/km CRICOS Provider Code 00301J Key Concept Map for Enzymes Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J Questions for learning Why is initial rate important when measuring enzyme activity? What does the Michaelis-Menten equation describe? Why is a Lineweaver-Burk plot often used in the study of enzymes? List at least three benefits to knowing Km Which kinetic parameters help give you an idea of an enzyme’s specificity? Explain the difference in operation between hexokinase and glucokinase What are the types of enzyme inhibition? Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J What do you do now? Read & Review Take a Quiz on the unit & get immediate Your text book and pdf and ilecture versions of this lecture FEEDBACK Login to Blackboard The quiz is another way to help you learn as it gives you feedback Do you have any unanswered questions? Attend a class and start the discussion; don’t be afraid to ask your lecturer Post your questions to the Blackboard discussion board to see if other students (or the lecturer) can help you. Proceed to the next unit! After you pass the quiz for this unit! Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J References and Bibliography Harper’s Biochemistry 32nd Ed. (2023). Mc Graw Hill Publishers Cornish-Bowden (1995) Fundamentals of Enzyme Kinetics. Revised Edition. Portland Press. Cornish-Bowden, A. (2013). The origins of enzyme kinetics. FEBS letters, 587(17), 2725-2730. Harvey, R.A., Ferrier, D.R., (2011) Lippincott's Illustrated Reviews: Biochemistry. 7th Edition. Lippincott Williams & Wilkins. Nelson and Cox (2000) Lehninger Principles of Biochemistry 3rd Edition.Worth Publishers Segel (1976) Biochemical Calculations 2nd Edition. John Wiley & Sons Faculty of Health Sciences | Curtin Medical School CRICOS Provider Code 00301J