Day 1: FACTORSINFLUENCINGENZYME ACTIVITY
Question:
How do factors such as temperature, pH, salinity, and concentration affect the rate of enzyme-facilitated reaction?
Knowledge Probe:
Enzymes are biological catalysts capable of speeding up chemical reactions by lowering activation energy. One benefit of enzyme catalysts is that the cell can carry out complex chemicalactivitiesatarelativelylowtemperature.
Mostenzymesareproteinsandtheir3-dimensionalshapeisimportanttotheircatalyticactivity. Twospecific regions ontheenzymestructure playanimportantroleincatalyticactivity:the activesiteandtheallostericsite.Theactivesiteistheareaoftheenzymewhichbindstothe substance(s) (substrate) andaidsinthechemicalreaction. The allostericsiteisinvolvedin formingtheproper3-dimensionalshapewhenlinkedwithspecificcofactors.Asa resultofthe uniquecharacteristicsofthesesites,enzymesarehighlyspecificintermsofthereactionsthey willcatalyzeandtheconditionunderwhichtheyworkbest.
In biochemicalreactionstheenzyme,combinesreversiblywithitsspecificsubstrate,to forman enzyme-substrate complex. Oneresult ofthistemporary unionisareduction inthe energy requiredtoactivatethereactionofthesubstratemoleculesothattheproductsofthereaction, areformed.
Thiscanbesummarizedintheequation:
Enzyme+ Substrate→Enzyme-SubstrateComplex→Enzyme+ Product
Notethattheenzymeisnotconsumedin thereactionandcanrecycletoworkwithadditional substratemolecules.Eachenzymeisspecificfora particularreactionbecauseits aminoacid sequenceisuniquewhichcausesittohaveaunique3-dimensionalstructure.Theactivesiteis theportionoftheenzymethatinteractswiththesubstrate,so thatanysubstancethatblocksor changestheshapeoftheactivesiteaffects theactivityoftheenzyme.
Inpractice,thisspecificity permitsonetomixapurifiedsubstrate withcrudepreparations of enzymethatmightcontainmanyothersubstancesandobtainaquantitativeassay(analysis)of theamountofenzymepresent.
Wewillbeworkinginthislabwitharepresentativeenzyme—
catalase. Catalase hasamolecular weight ofapproximately
240,000daltonsand contains4 polypeptidechains,each composed of more than 500 amino acid monomers. This enzymeoccursuniversallyinaerobicorganisms.Onefunction of catalasewithincellsis topreventthe accumulationof toxic levelsof hydrogenperoxide(H2O2) formedasaby-productof metabolicprocesses.Catalasemightalsotakepartinsomeof themanyoxidationreactionsgoingoninallcells.
The primary reaction catalyzed by catalase is the decompositionofH2O2toformwaterandoxygen.
2H2O2→2H2O+O2(gas)
In theabsenceofcatalase,thisreactionoccursspontaneously, butveryslowly.Catalasespeedsupthereactionconsiderably. Inthisexperiment,arateforthisreactionwillbedetermined.
Thecatalasethatworksinliver andinredbloodcells.
Muchcanbelearnedaboutenzymes bystudying thekinetics (changes inrate)ofenzyme- catalyzedreactions.Forexample,itispossibletomeasuretheamountofproductformed,or the amountofsubstrateused,fromthemomentthereactantsarebroughttogetheruntilthereaction hasstopped.
Solet’slookatahypotheticalexample:
Anenzymeanditssubstratearemixedin areactionvessel.If theamountofproductformedis measuredat30secondintervalsandthisquantityplottedonagraph,acurveliketheonein Figure1isobtained:
Figure1.EnzymeActivity
40
Product 30
(µmoles)20
10
0 1 2 3 4 5 6 7 8
Time (minutes)
Observethesolidlineforthisreaction.Attime0 thereisnoproduct.After30 seconds,5 µmoles havebeenformed;after1minute,10;after2minutes20.Therateofreactioncouldbegivenas
10µmolesofproductformedperminuteforthisinitialperiod.Note,however,thatbythe3rdand
4th minutes onlyabout 5additional µmoles ofproduct have beenformed. During thefirst3 minutes,therateisconstant.Fromthe3rdminutethroughthe8thminute,therateischanging— it isslowingdown.Foreachsuccessiveminuteafterthefirst3minutes,theamountofproduct formedinthatintervalislessthanintheprecedingminute.Fromthe7thminuteonward,the
reactionrateisveryslow.
Inthecomparisonofkineticsofonereactionwithanother,acommonreferencepointisneeded. For example, suppose you wanted tocompare the effectiveness ofcatalase obtained from potatowiththatofcatalaseobtainedfromliver.Wouldyouwanttocomparethetworeactions duringthefirstfewminuteswhentherateisconstantorlaterwhentheratesarechanging?
Answer:Itisbesttocomparethereactionswhentheratesareconstant.
In the first few minutes of an enzymatic reaction such as this, the number of substrate moleculesisusuallyso largecomparedto thenumberofenzymemoleculesthattheenzymeis constantlyhavingsuccessfulcollisions withsubstrate. Therefore, duringthisearlyperiod,the enzymeisactingonsubstratemoleculesata constantrate (asfastasitcan).Theslopeofa graphedlineduringthisearly periodiscalledtheinitialvelocityofthereaction. Theinitial velocity(orrate)ofanyenzyme-catalyzed reactionisdeterminedbythecharacteristics ofthe enzymemolecule. Itisalwaysthesameforaspecific enzymeanditssubstrate aslongas temperatureandpHareconstantandthesubstrateispresentinexcess.
Theinitialrateof thereaction,thereforeis theslopeof thelinearportionof thecurve.To determine a rate, pick any two points on the straight-line portion of the curve. Divide thedifferencein theamountofproductformedbetweenthesetwopointsbythedifferencein time betweenthem.Theresultwillbe therateofthereactionwhich,ifproperlycalculated,canbe expressedasµmolesofproduct/second.Thisequationis:
µmoles2-µmole1
t2–t1
InthegraphshownasFigure1:
30–20 / = / 10 / = / 0.17 µmoles/second180–120 / 60
Therateofachemicalreactionmaybestudiedinanumberofways,includingthefollowing:
1. Measuringtherateofdisappearanceofsubstrate,inthisexample,H2O2
2. Measuringtherateofappearance ofproduct,inthisexample,O2,whichis givenoffasagas.
3. Measuringtheheatreleased(orabsorbed)duringthereaction.
Inthisexperiment,thedisappearanceofsubstrate,H2O2,andthegenerationofproduct,O2,aremeasured.
Materials:
Obtainthefollowingmaterials:
•50mLbeakercontainingfresh catalase(yeast)solution
• reactionchamber
• ringstandclamp
• 10mLgraduatedcylinder
• 100mLgraduatedcylinder
• 3%hydrogenperoxide(H2O2)
• pan(waterbath)
• pipette
• hotplate
• ice
• thermometer
• boiledcatalase
• buffersofvaryingpH:4,7,10
• distilledwater
• balance
• NaCl(salt)
1. Workasalabgroupof3-4members. Eachlabgroupwillcomplete PartAand PartBofthe lab.Yourteacherwillassignonanadditionalactivitytoeachgroupform PartsC,D,EF.
2. Atyourlabbenchyouwillfindaroundvialwitharubberstoppertop.Thisiscalledthe reactionchamber. Youwillalsofinda100mLgraduatedcylinder,ringstandandclamp, andaplasticpanwhichwillbeusedasawaterbath.Allofthisequipment needstobe assembledintoourexperimentalapparatus,asdescribedandillustratedbelow.
3. Fillthepan3/4fulloftapwater.Allowthewatertocometoroomtemperature.
4. Submerge the100mLgraduatedcylindertofillitwithwater.Turnthegraduatedcylinder upsidedown,keepingtheopenendunderwater,soastokeepitfilledwithwater.Suspend itupsidedownintheclampontheringstand.Adjusttheheightoftheclamponthering standsotheopenendofthegraduatedcylinderisabout3cmabovethebottomofthepan. Seediagrambelow.
5. Placeathermometerinthepanandrecordthetemperature
ofthewater, duringPartAofthelab.
°C
6. Whenallsectionsofthelabarecomplete,sharethedatawiththeclassfromyourgroup’s section.Eachpersonmustplotthedataforallpartsofthelabonhis/herowngraphpaper.
FACTORSINFLUENCINGTHE ACTIVITYOFCATALASE
Severalenzymaticvariableswillbeexaminedin thislab.Youwillbeusingtheproteinenzyme, catalase.Catalaseisfoundinmostcells,eveninsingle-celledeukaryoteslikeyeast.Inthislab, youwillextractcatalasefroma yeastsolutionandtestitscatalyticeffectivenessonhydrogen peroxide.Catalasespeedsup thebreakdownofperoxideswhichmayformduringrespiration (metabolicenergyproduction).Thisbreakdownpreventstheperoxidefrom causingunwanted oxidationofimportantbiomolecules:
2H2O2→2H2O+O2(gas)
Wewill measureenzymeactivity bymeasuringthegenerationof oxygengas—a productinthereaction.
PartA.TheTimeCourseofEnzymeActivity
1. Setuptheexperimentalapparatusasillustratedanddescribedabove.
2. Obtainareactionchamber.
3. Obtainabottleof3%hydrogenperoxide(H2O2)solutionanda10mLgraduatedcylinder.
4. Obtainasmallamount ofstockcatalase(yeast) solutionina50mLbeaker. Youwill need1.0mLofyeastsolutionforeachtrial.Whenyouareready,youwilladdit to thevial withaplasticpipette.
5. Pour10mLofhydrogenperoxide(H2O2)intothereactionchamber.Pipettein 1.0mLof stockcatalasesolution(yeastsolution)andIMMEDIATELYstopperthereaction chambertightly,submergeitinthewaterbathandplacetheplastictubingintothe bottomofthegraduatedcylinder,soallthebubblesformedinthereactionchamberare capturedbytheinvertedgraduatedcylinder.
6. Measurethegaslevelsinthegraduatedcylinderat 30-secondintervalsfor5minutes.
(In the observation section): Recordthelevelsinadatatableofyourowndesign.
7. (In the data analysis section): Plotthedataonagraph.Don’tforgettolabelyouraxes andtitleyourgraph.
PartB.TheEffectofEnzymeConcentrationon EnzymeActivity
1. Repeattheexperiment fromPartA,using3differentlevelsofenzymeconcentration:
75%,50%,and25%concentrationofenzymesolution.Youmayeasilydothisbyusing thefollowingprocedures:
a. 75%concentration:FollowtheprocedurefromPartA,butuse 0.75mLcatalase
solutioninthereactionchamber,insteadof1.0mL.
b. 50%concentration:FollowtheprocedurefromPartA,butuse 0.50mLcatalase
solutioninthereactionchamber,insteadof1.0mL.
c. 25%concentration:FollowtheprocedurefromPartA,butuse 0.25mLcatalase
solutioninthereactionchamber,insteadof1.0mL.
2. (In the observation section): Recordalldatainadatatableofyourowndesign.
3. (In the data analysis section): PlotthedataonthesamegraphasPartA.Don’tforgettoclearlylabeltheenzyme concentrationsonyourplottedlines.
Day 2: FACTORSINFLUENCINGENZYME ACTIVITY
PartC.TheEffectofTemperatureonEnzymeActivity
Design how to test the effects of temperature on enzyme activity. How many trials will you need? What temperatures will you use and why? Write out your investigative plan.
PartD.TheEffectofpH onEnzymeActivity
Design how to test the effects of pH on enzyme activity. How many trials will you need? What pH levels will you use and why? Write out your investigative plan.
PartE.TheEffectofSubstrateConcentrationonEnzymeActivity
Design how to test the effects of substrate concentration on enzyme activity. How many trials will you need? What enzyme concentrations will you use and why? Write out your investigative plan.
Part F.TheEffectofIonicConcentrationonEnzymeActivity
Here you will use different concentrations of salt solution. Your strongest solution will be 10% NaCl. Design how to test the effects of ionic concentrations on enzyme activity. How many trials will you need? What ionic concentrations will you use and why? Write out your investigative plan.
Observation:
Data Analysis:
Explanation:
→Claim-- Does the claim answer the investigation question?
→Evidence-- Does the selected evidence support the claim?
→Reasoning-- Does the reasoning link the claim and evidence?
Does the reasoning justify why the evidence supports the claim?
Does the reasoning make a strong argument?
Does the reasoning consider alternative explanations?
(Use one group as an example first)--Have partner groups present and defend to each other and then come up with a recommendation for the whole class.
Evaluation—
a.What are the sources of error?
b.What would you do differently next time?
c.How confident are you in your results? (See Confidence Chart) What could be alternative explanations for my results?
d.What surprised you?
e.What would your prediction be if you conducted this investigation again?
f. What question would you like to pursue next?
→Application: How do I use what I learned?