ppt enzyme kinetics (2006)

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  • Enzyme KineticsLab C1Two periodsPages 73-104

  • Protein ChemistryThis begins a 6 day journey into the field of protein chemistryYou will learn a set of basic tools and protocols which will be important in the successful outcome. You have practicedMeasurement accuracySpectrophotometryRelationship between concentration and absorbanceToday we will do a basic experiment in enzymology which will prepare you for a protein purification.

  • EnzymesLiving organisms must be able to carry out events which are thermodynamically very unfavorableBreak and form covalent bondsMove large structuresEffect three dimensional structureRegulate gene expressionDo so through use of Enzymes

  • Effect of enzymesA bag of sugar can be stored for years with very little conversion to CO2 and H2OThis conversion is basic to lifeThis common biological reaction can take place without enzyme catalysisWill take 750,000,000 yearsEven improvement of a factor of 1,000 would be goodOnly 750,000 yearsLiving systems would be impossible

    With enzyme 22 milliseconds

  • CatalysisCarried out by very highly specialized class of proteins: EnzymesSpecialized to perform specific chemical reactionsSpecialized to work in specific environments

  • EnzymesHave immense importance in a number of fields.Genetic diseases are frequently defects in enzymes or increased/decreased levels of enzymesImportant diagnostic toolsDrugs exert effects by interacting with enzymesMAO inhibitorsUsed in food processing and in chemical industryEnzyme inhibitors are a foundation of biological weapons

  • Enzymes A major aspect of experimental biochemistry is the purification and characterization of proteins that are enzymesChemical characterizationPhysical characterization

  • In the next six laboratoriesYou will go through the basic protocols that are used to purify and characterize catalytic proteinsThe basic procedures are ones which you will use the rest of your career if you choose a career in biochemistry, molecular biology, biophysics, biochemical genetics, pharmacology, cell biology, etc

  • KineticsIs the science that describes the properties of a chemical reaction including those mediated by enzymes (catalysis)Measures changes in the concentration of substrate and/or products of a reaction with time to determine the velocity of the reactionMeasures the effects of concentration, temperature, pH etc. to characterize the properties of the enzyme catalyzing the reaction

  • Stickase From Lehninger; third edition

  • Enzyme KineticsAn approach to understanding the mechanism of action of enzymesAn approach to understanding how mutations may effect functionAn approach to understanding how changes in the physical and chemical environments change function

  • Rate Constant: kA BVelocity of Rx

    V=[B]/tV=-[A]/tV=[B]/t = -[A]/t = k[A]

    Units are quantity/unit timee.g. Moles/SecondLarge k rapid RxSmall k slow Rx

  • CatalysisSimple reaction A [s] B [P]E + S ES E + P

    K2 also known as kcat

    At steady state

    [ES] = (k1/k-1 + k2) [E] [S]k-1k2k1

  • km: A ratio of Rate constantspage 80-81(Info Box 5)[ES] = (k1/k-1 + k2) [E] [S]

    km= k-1 + k2/ k1

    Km =Michaelis constant

  • Initial velocity VoWhen enzyme is mixed with high concentration of substrate [S] reaction goes rapidly to steady state.Does not allow characterizationUse low starting [S] and increaseHold [enzyme] constantMeasure initial rate of reaction, Vo as [S] increasesUntil rate becomes constant: approaches Vmax

  • Effect of [Substrate]

  • Effect of [substrate] on RX Velocity

  • Michaelis-Menten EquationV0 = Vmax [S]

    Km +[S]

  • Lineweaver-Burk PlotUnits of Km are concentration

  • Can calculate KmOne of the most important descriptive terms in all of biology

  • Alcohol Dehydrogenase: ADHCH3CH2OH + NAD+ CH3CH2O + H+

    +NADH

    Catalyses conversion of ethanol to aldehyde using co-enzyme NAD+

    NAD+ oxidized to NADH reduced

  • NAD+

  • NAD+ to NADHAbsorbs at 340

  • NADH

  • Reaction is complexADH +ALC ADH-ALCADH + NAD ADH-NADADH-NAD +ALC ADH-NAD-ALC

    We are not looking at this

  • Alcohol DehydrogenaseCH3CH2OH + NAD+ CH3CH2O + H++ NADHWe will measure the forward Rx (k 2)as increased absorbance at 340. Only NADH absorbs at this wave length (page 70)

    Will find the assay conditions which produce max activity and calculate Km

  • WHAT ARE WE MEASURING ?Production of NADH

    NAD+ NADHWavelength shiftDepends on participation of Alcohol and ADHHow can you do thisEnsure that NAD is not a rate limiting component.

    [NAD] constant and high[ADH] constant [ETOH] low and increasing

  • Measure Vo with increasing [S] Re-plot these data in the double-reciprocal Lineweaver-Burk plot

    Remember Vo= NADH/ Time.

  • This Lab and Next LabPart one Kinetic Curve (Figure C.1-5), V0 Lineweaver-Burk (Figure C.1-6) page 86-88Determine basic properties of enzyme KMPart two Page 89-92Effects of temperature and pHReport requirements: Page 102-104.

  • Experiment 1: Page 86&76

    Kinetic curve. Add enzyme

    19.cdx

  • Experiment 2 Page 87-88Determine Km and VmaxPipetting accuracy and timing is criticalClean cuvetteCan check clean by adding all components except ADH and placing in spectrophotometerAbsorbance should not change with time

  • Data tablePage 87Km

    Table C.1-1. ____Assay #Water (ml)BufferEthanol[S]NAD+ADHV1/V1/[S]10.0000.7002.1000.1000.10020.6000.7001.5000.1000.10031.1000.7001.0000.1000.10041.6000.7000.5000.1000.10051.9000.7000.2000.1000.10062.0000.7000.1000.1000.10072.0500.7000.0500.1000.10082.0800.7000.0200.1000.10092.0900.7000.0100.1000.100102.0950.7000.0050.1000.100

  • Be careful 15 sec and 45 secRead same and low = too little substrateDidnt add enzymeRead same and highReaction is over Contaminated one of your solutions with enzymeDid not clean cuvette from previous assay

  • Initial Velocity (Vo page 85)

  • Sample dataKinetic curve Figure C.1-5Lineweaver-Burke Plot Figure C.1.6

  • This Lab2 Lab periodsPre Labs 6 pointsLab Report 20 points

  • Clean up and Check outPage 101-102Return pipetters to rackCheck that you have not left cuvette in specClean any spill in specClean & rinse the cuvetteClean and rinse test tubesThrow all waste in trash

  • Next timeExamine the effects of:TemperaturepH

  • Next Exercise

    Effects of Temp, pH and Enzyme concentration. Page 89-92Read carefully Factors that affect catalysis (Page 93-101)prior to coming to lab.Lab report on Enzyme Kinetics due at start of protein purificationRemember to find the Km of another enzyme and compare it to ADH

  • Temperature Dependence page 94

    20.cdx

  • Effect of pH page 99

    21.cdx

  • Extra Credit for this Lab5 pointsAt lower temperatures the kinetic rate change with temperature demonstrates Arrhenius behaviorArrhenius Plot: Plot log Vo versus 1/T degrees Kelvin, determine activation energyShould result in a straight lineSlope = Ea (activation energy)/ R (Gas constant 1.9872041(18)103 Kcal/molhttps://www.youtube.com/watch?v=Brf-_oyLFGwShows how to calculate using Xcel

    30.cdx

  • Arrhenius plotSlope = _Ea/RR= 1.9872041(18)103 Kcal/mol

  • Kinetics Write UpSee report outline Page 102

    Remember describe what happened in your experiment

  • In your reportEmphasize what you have learned about the enzyme alcohol dehydrogenaseIts maximum velocityIts ability to produce product at steady stateIts KmHow efficient is ADH in forming the ES complexHow does it compare to other enzymesIts optimal pHIn what environment does it function bestIts optimal temperatureIts activation energy (if calculated)How many Kcals or Joules are required to produce a mole of ethyl aldehyde

  • Look at the family of Dehydrogenaseshttp://en.wikipedia.org/wiki/DehydrogenaseWhat generalizations can you make regarding your observations on ADH and the properties of these other enzymes.

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