BIOC 460, Spring 2008LEC 12, Enzymes - Inhibition 1Lecture 12Enzymes: InhibitionReading: Berg, Tymoczko & Stryer, 6th ed., Chapter 8, pp.225-236Problems: pp. 238-239, chapter 8, #1, 2, 4a,b, 5a,b, 7, 10Jmol structure: cyclooxygenase/non-steroidal anti-inflammatory drugs:http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/cox12/cox121.htmKey Concepts• Km and Vmax can be determined from double reciprocal plots (1/Vo vs. 1/[S]).• Enzyme inhibitors: compounds that reduce velocity of enzyme-catalyzedreactions.• 2 types: reversible and irreversible• reversible inhibitors– competitive: inhibitor (I) increases Km but has no effect on Vmax.– uncompetitive: I decreases both Km and Vmax by same factor.– pure noncompetitive: I decreases Vmax but has no effect on Km.– can distinguish different types of reversible inhibitors using double reciprocal plots (1/Vo vs. 1/[S]) in absence of I and in presence of different concentrations of I.• Irreversible inhibitors – cause irreversible (generally covalent) modification of the enzyme, inactivating it. – several types: • group-specific chemical modifying reagents that would react with certain types of functional groups on many different enzymes• substrate analogs with a reactive group on them (so more specific for one enzyme)• "suicide" substrates (mechanism-based inhibitors): not reactive until the specific chemical mechanism of their target enzyme makes them "kill" (covalently modify) the active site they're in.BIOC 460, Spring 2008LEC 12, Enzymes - Inhibition 2Key Concepts, continued• Both reversible and irreversible inhibitors very helpful for:– providing information about shape of active site and types of amino acidside chains there– working out enzyme mechanisms– providing info about control of metabolic pathways– design of drugs Learning Objectives• Terminology: double reciprocal plot, reversible inhibition (competitive, pure noncompetitive, uncompetitive), irreversible inhibition, affinity label, transition state analog, suicide inhibition (mechanism-based inhibitor)• Given a hyperbolic Vo/Vmax vs. [S] plot, explain the meaning of the ratio Vo/Vmax in terms of occupied active site concentration and total active site concentration, and find Km directly from the graph, including its units.• Given a Lineweaver-Burk plot (1/Vo vs. 1/[S]), find/calculate Vmax and Km from the graph.Learning Objectives, continued• Explain competitive, uncompetitive, and pure noncompetitive inhibitionin terms of a diagram of linked reaction equilibria for formation of ES,EI, and (if it can form) EIS. (See Berg et al., Figs. 8-17, 8-18, 8-19.)– Explain how these 3 types of inhibition can be distinguished fromeach other graphically:a) on a Vo vs. [S] plot, andb) on a double reciprocal (Lineweaver-Burk) plot.• What is the effect of a competitive inhibitor on Km and on Vmax(compared to the values in absence of inhibitor)?• What is the effect of an uncompetitive inhibitor on Km and on Vmax(compared to the values in absence of inhibitor)?• What is the effect of a pure noncompetitive inhibitor on Km and onVmax (compared to the values in absence of inhibitor)?• Why is ethanol used as an antidote for ethylene glycol (antifreeze)poisoning?• What type of inhibitor is pencillin?• Give some other specific examples of reversible and irreversibleenzyme inhibition.BIOC 460, Spring 2008LEC 12, Enzymes - Inhibition 3Graphical Determination of Km and Vmax• Enzyme kinetics (experiments measuring Vo as a function of [S] todetermine Vmax and Km)– can use computer with programs to fit data to Michaelis-Mentenequation, extracting Km and Vmax from the data.– can’t extrapolate "by hand" on Michaelis-Menten Vo vs. [S]hyperbolic plot to get accurate Vmax and Km values– simple solution without a computer: use linear version, double reciprocal plot (Lineweaver-Burk Plot)– Take reciprocal of both sides of M-M Equation and rearrange to get Lineweaver-Burk equation:Lineweaver Burk Plot (double reciprocal plot)• Equation of a straight line: y = mx + b• x–intercept = – 1/Km• NOTE: x-intercept is a negative number, which itself is the negative of the reciprocal of Km• (–) (–) = (+)• Km is always a positive number (the substrate concentration at which V = 1/2 Vmax). • y-intercept = 1/Vmax • slope = Km / Vmax Lineweaver-Burk Plot (double reciprocal plot, 1/Vo vs. 1/[S])Berg et al., Fig. 8-13BIOC 460, Spring 2008LEC 12, Enzymes - Inhibition 4Lineweaver Burk Plot (double reciprocal plot)• Equation of a straight line: y = mx + b• x–intercept = – 1/Km• y-intercept = 1/Vmax • slope = Km / Vmax Lineweaver-Burk Plot (double reciprocal plot, 1/Vo vs. 1/[S])• What if x-intercept = – 2 x 104 M–1? (a reciprocal concentration, extrapolated back to negative values to get intercept) – 1/ Km = – 2 x 104 M–1–1 1–2III• What is Km? • Km = – 1/(–2 x 104 M–1) = + 0.5 x 10–4 M (or 5 x 10–5 M) • Note POSITIVE value! (Can Km ever have a negative value?)Enzyme Inhibitors• reversible (rapid binding/release from enzyme in an equilibrium) or• irreversible (very tightly bound to enzyme, either covalently ornoncovalently, but effectively don't come off)• Reversible inhibitors– type of inhibition diagnosed by effect of inhibitor on Km and Vmax– effects/"diagnosis" obvious on double reciprocal plot.– basis for drugs' actions– research tools in figuring out enzyme chemical catalytic mechanisms• Reversible Inhibition:–competitive–uncompetitive, or–noncompetitive– defined operationally by their effects on enzyme kinetic parameters, Km and/or Vmax.BIOC 460, Spring 2008LEC 12, Enzymes - Inhibition 5Reversible Enzyme Inhibitors, continued• Decreases Km and reduces Vmax by same factor so slope of 1/Vo vs. 1/[S] doesn’t change• binds only to ES complex• Has no effect on Km (no effect on S binding)• reduces Vmax (reduces kcat)“Pure”Berg et al. Fig. 8-15ES Complex• Prevents S from binding, so increases Km• no effect on VmaxCompetitive Inhibition• Enzyme can bind either substrate or inhibitor, but not both• Either– Inhibitor binds in same site as S, or– (more rarely) inhibitor binds to different site, causing conformationalchange in active site so substrate can't bind.• Enzyme active site can be free (neither ligand bound, E) or have Sbound (ES complex) or
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