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BIOL121 NAME: Exam1 10/02/09 PENNID#: (PleasePRINTmiddle8digitsonly)

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INSTRUCTIONS: 1. Donotopenthisexamuntilinstructedtodoso. 2. Thisisaclosedbookexam.WewillobservetheCODEOFACADEMICINTEGRITY. Pleasesignthepledgebelow. 3. Writeyournameoneverypage. 4. WriteyouranswersinPEN,notpencil,unlessyouarepreparedtowaiveyour righttomakearegraderequest. 5. Makesureyouanswerallofthequestions. 6. PleasewriteLEGIBLY! 7. Notethatcorruptionofacorrectanswerwitherroneousorsuperfluoustextwill resultinlesscredit. 8. Pleaseenteryouranswerstotheshortanswerquestionsinthespaceprovided donotwriteonthefacepageortheothersideofthepage. 9. Time:youhave50minutestocompletethisexam. IpledgemyhonorthatIhaveupheldtheCODEOFACADEMICINTEGRITYoftheUniversity ofPennsylvaniaincompletingthisexamination

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Points

BIOL121 Exam1 10/02/09

NAME:

MULTIPLECHOICEcirclethecorrectanswer(s) PLEASENOTETHATMORETHANONEANSWERMAYBECORRECT VERYSHORTANSWERQUESTIONSanswerthequestionsin2or3sentences 1) Iftheaminoacidshownbelowwasburiedintheinteriorofasolubleglobular protein,andassumedthechargestateshownforthesidechainunder physiologicalconditions,itwouldmostlikely(butnotnecessarily)befoundnear aminoacid: a(n)

(3 pts)

A. B. C. D. E. F.

neutral,polar neutral,nonpolar basic acidic sulfurcontaining aromatic

2)

(3 pts)

Theconformationofpolypeptidechainsisprimarilystabilizedby: A. hydrogenbondsbetweentheconstituentatomsofmainchainpeptide bonds. B. electrostaticinteractionsbetweenRgroups. C. hydrophobicinteractionsbetweencarbonsofthemainchain. D. hydrogenbondingbetweentheRgroups. E. hydrophobicinteractionsbetweenRgroupsandthesolvent,waterinthis case.

BIOL121 Exam1 10/02/09

NAME:

3)

4)

(3 pts)

Whattypesofinteractionswouldyouexpecttobecapableofpromotingthe associationofperipheralmembraneproteinswithintegralmembraneproteins? A. Hbonding B. Hydrophobicinteractions C. Electrostaticinteractions D. Disulfidebonds E. A,CandD F. A,BandC G. A,B,CandD

Oftheaminoacidsshownbelowwhichwouldyouexpecttopromotethe associationofintegralmembraneproteinswiththehydrocarbonphaseofthe phospholipidbilayerofamembrane?[Assumethatthesidechainsofthoseamino acidsthatarecapableofundergoingprotonationordissociationatatypical physiologicalpHofapproximately7doso].

(4 pts)

A. B. C. D. E.

AandD BandE CandF A,B,andC D,E,andF

BIOL121 Exam1 10/02/09

NAME:

5) Humanbloodserumcontainsaclassofenzymesknownasacidphosphatases whichhydrolyzeorganicphosphatesunderslightlyacidicconditions(pH5.0): ROPO32+H2OROH+HPO42 Acidphosphatasesareproducedbyawiderangeofcellsincludingerythrocytes, andthoseoftheliver,kidney,spleenandprostategland.Theenzymeproducedby theprostateglandisofclinicaldiagnosticvaluebecauseanincreaseinitslevelsin thebloodcanbeindicativeofprostatecancer.Thephosphatasefromtheprostate glandisstronglyinhibitedbytartrateanion(adicarboxylicacid)butacid phosphatasesfromothercelltypesarenot.Howmightthisinformationbe exploitedforthedevelopmentofaspecificprocedureformeasuringtheactivity prostaticacidphosphataseactivityinhumanbloodserum?Ifyouweretouseits catalyticactivityasanindexoftheamountofthisenzymeinbloodserumwhat precautionswouldyouhavetotake?
(8 pts)

The activity of the prostatic acid phosphatase would correspond to the total acid phosphatase activity of the blood sample minus the acid phosphatase activity measured in the presence of a sufficiently high concentration of tartrate to completely inhibit the prostatic enzyme. If catalytic activity is to be a reliable index of the amount of the enzyme in blood serum it is not only crucial that sufficient tartrate is added to the sample when enumerating the contribution of the prostatic enzyme to total acid phosphatase activity but that the concentration of substrate (an organic phosphate) added is sufficient to ensure that the enzyme is operating at close to maximal activity (Vmax) such that reaction velocity, v, is directly proportional to prostatic enzyme concentration: v = k[E]t where k is a proportionality constant and [E]t is the total concentration of the prostatic enzyme.

6) Youhaveidentifiedanorganismthatcanmaintainthefluidityofitsplasma membraneevenatverylowtemperatures.Youanalyzeplasmamembrane samplesfromthisorganismaftergrowthatmoderatetemperaturesandafter growthatlowtemperaturesanddeterminetheircontentsofsaturatedandcis unsaturatedphospholipids.Whatwouldyouexpecttofind? A. Nochangeintheratioofsaturatedtounsaturatedphospholipidsatlow temperatures B. Anincreaseintheratioofsaturatedtounsaturatedphospholipidsatlow temperatures. C. Anincreaseintheratioofunsaturatedtosaturatedphospholipidsatlow temperatures Howwouldyourexpectationschange,ifatall,iftheunsaturatedphospholipidsin questionwereinthetransratherthanthecisform?Brieflyexplainyourreasoning.
(4 pts) (3 pts)

cis double bonds in the fatty acyl hydrocarbon chains of phosphopholipids introduce

kinks that decrease the ease with which phospholipids associate with each other to form a crystal lattice (undergo solidification) at lower temperatures. trans double bonds do not have this effect because they do not introduce kinks into the fatty acyl hydrocarbon chains; their behavior is more like that of fatty acyl substituents possessing saturated hydrocarbon chains.

BIOL121 Exam1 10/02/09

NAME:

7) TheuptakeofDglucosefromtheintestinallumen(fromthecontentsofthegut) bytheNa+coupledtransporterassociatedwiththeluminal(apical)membraneof thecellsoftheintestinalepithelium: A. isanexampleofantiport. B. iscapableofmediatingtheaccumulationofglucoseevenwhen [Na+]extracellular=[Na+]intracellularprovidingthemembranepotentialisnegative (insidenegative). C. iscapableofmediatingtheaccumulationofglucoseevenwhenthe membranepotentialispositive(insidepositive)providingthat [Na+]extracellular=[Na+]intracellular. D. isonlycapableofmediatingtheaccumulationofglucosewhenthe extracellularconcentrationofNa+isgreaterthantheintracellular concentration.
(4 pts)

8)

(4 pts)

Whichofthefollowingstatementsaboutallostericenzymesandtheirregulation is/areCORRECT A. Theyareoftenfoundatkeystepsinmetabolicpathwaysandundergo feedbackregulation. B. Theyusuallyexhibitcooperativekinetics. C. Thebindingofanallostericactivatorincreasestheactivityoftheenzyme atallsubstrateconcentrations. D. Allostericactivatorsusuallyincreasetheconcentrationofsubstrate requiredfor50%saturationoftheenzymebyenhancingthesigmoidicity ofvversus[S]plots. E. Theeffectsofactivatorsandinhibitorsonthecooperativeinteractions betweensubstratebindingsubunitspermitchangesinvevenwhenthe concentrationofsubstrateremainsthesame. F. Ifacompetitiveinhibitorisaddedtoasolutioncontaininganallosteric enzyme,theratioofenzymemoleculesintheactiveconformationto thoseintheinactiveconformationwould(probably)increase.

9) Fromwhatyouknowoftheenergyyieldfromtheanaerobicversusaerobic metabolismofglucose,whatwouldyoupredictforacellthatiscapableof operatinganaerobicallyoraerobicallywhenthecellistransferredfromanaerobic toaerobicconditions(ifthecellistoharvestthesameamountofenergyperunit timeunderbothconditions)?Assumethatthesupplyofglucoseisplentiful: A. Agreaterthan16foldincreaseintherateofglucoseconsumption. B. Anapproximately2to4foldincreaseintherateofglucoseconsumption. C. Agreaterthan16folddecreaseintherateofglucoseconsumption. D. Anapproximately2to4folddecreaseintherateofglucose consumption. E. Nochangeintherateofglucoseconsumption.
(4 pts)

BIOL121 Exam1 10/02/09

NAME:

10)RespiratoryCO2,theCO2youareexpiringwhilereadingthisquestion,isderived from: A. thesamereactionastheCO2producedbybrewersyeastgrownunder anaerobicconditions. B. theoxidationoflactatetopyruvate. C. theTCAcycle. D. theoxidativedecarboxylationofpyruvate. E. therespiratoryelectrontransportchain. F. oxidativephosphorylation.


(4 pts)

11)Supposethatyouhavebeengivenasuspensionofbovineheartmitochondriathat ismediatingthesteadystateoxidationofanNADcoupledsubstrateinamedium containinganexcessofoxidizablesubstrate,NAD+,ADP,Pi,andO2.Whatwould youexpecttoobserveifanuncoupler,forexample2,4dinitrophenol(2,4DNP), wereaddedtothissuspension? A. Adecreaseintherateofsubstrateoxidation. B. Anincreaseintherateofsubstrateoxidation. C. AdecreaseintherateofNADHoxidation. D. AnincreaseintherateofNADHoxidation. E. AdecreaseintherateofATPsynthesisfromADPandPi. F. AnincreaseintherateofATPsynthesisfromADPandPi. G. AdecreaseintherateofO2consumption. H. AnincreaseintherateofO2consumption. (4 pts)

SHORTANSWERQUESTIONS ONLYAFEWSENTENCESATMOSTARENEEDEDFORACOMPLETEANSWERTO EACHSECTION Pleaseenteryouranswersinthespaceprovidedtrytoavoidwritingonthefacepage ortheothersideofthepage. 12) Allostericenzymesaremadeupofmultiplesubunitsandsomeoftheseenzymes existinonlytwoconformationalstates,designatedRorT(forRelaxedand Tense).Assumeforthetetramericallostericenzymeinquestionthatsubstrate (S)onlybindstotheRstate.Intheabsenceofligand(substrate,allosteric inhibitorsorallostericactivators),thetwostates(RorT)ofsuchanallosteric proteinareinequilibrium:RT.TheequilibriumconstantL(definedasL=T/R) isusuallyverylarge;thatis,theamountoftheproteinintheTstateismuch greaterthantheamountintheRconformation. ExplainwhathappenstotheequilibriumbetweenRandTasincreasingamountsof substrateareaddedtoasolutioncontainingtheenzyme. (5 pts)
The equilibrium between the T and R conformational states would be displaced in favor of the R conformation as increasing amounts of substrate are added to the solution containing enzyme. If the enzyme is in dynamic equilibrium between the T and R states and substrate only binds to the R state, the addition of increasing amounts of substrate to the solution containing enzyme would serve to trap increasing amounts of the enzyme in the R state.

BIOL121 Exam1 10/02/09

NAME:

Explainwhyaplotofreactionvelocity(v)versussubstrateconcentration([S])for anenzymethatshowsthisbehaviordoesnotobeysimpleMichaelisMenten kineticsbutisinsteadsigmoid(Sshaped). (4 pts)


Sigmoid v versus [S] plots are obtained when an enzyme undergoes conformational changes of this type because the binding of substrate to the active site on one subunit promotes the binding of further substrate molecules to the other active sites on the other subunits. As is the case for most, if not all, allosteric enzymes, the binding of substrate to one site can affect the properties of the other sites in the same enzyme molecule because the individual subunits in the enzyme are capable of cooperative interactions whereby a conformational change in one subunit is transmitted to adjacent subunits. At low substrate concentrations most of the enzyme is in the inactive T state but as more of the enzyme is trapped in the R state by substrate binding more and more enzyme molecules assume the active R state. It is as if with increase in [S] the enzyme switches from a very low (or no) affinity form to a high affinity form.

BIOL121 Exam1 10/02/09

NAME:

ExplainwhathappenstotheequilibriumbetweenRandTwhenanallosteric activator(positivemodulator)isaddedtotheenzyme;explainwhathappens whenanallostericinhibitor(negativemodulator)isadded. (5 pts)


Allosteric activators bind to sites distinct from the substrate binding sites of the enzyme (regulatory sites) and trap the enzyme in the R conformation; the conformation with high affinity for substrate. In so doing they decrease the apparent k0.5 for substrate binding of the enzyme population as a whole. Allosteric inhibitors bind to sites distinct from the substrate binding sites of the enzyme (regulatory sites) and trap the enzyme in the T conformation; the conformation with low (or no) affinity for substrate. In so doing they increase the apparent k0.5 for substrate binding of the enzyme population as a whole. Allosteric activators preferentially bind the R conformation and bias the R T equilibrium in favor of R; allosteric inhibitors preferentially bind the T conformation and bias the R T equilibrium in favor of T.

Onthebasisofwhatyouknowaboutallostericenzymes,whatwouldyoupredict wouldhappentotherelationshipbetweenvand[S]iftheindividualsubunitsof thetetramerdepictedonthepreviouspageweredissociatedfromeach,i.e.the enzymehaddissociatedintoitsconstituentmonomers.Assumethatthe monomericformoftheenzymehasatleastsomecatalyticactivity. (6 pts)


If the monomeric form of this enzyme indeed has catalytic activity, it is likely that dissociation of the individual subunits from each other would yield an enzyme preparation that exhibits simple Michaelis-Menten-type kinetics rather than sigmoid kinetics. Cooperative interactions of the type described above arise when an enzyme is multimeric and the conformational state of one subunit influences the conformational states of the other subunits. If the enzyme is no longer multimeric it is no longer able to engage in such cooperative interactions. In short, conversion of the enzyme from a multimeric to monomeric state would change the dependence of v on [S] from a sigmoid relationship to a (rectangular) hyperbolic relationship.

BIOL121 Exam1 10/02/09

NAME:

13)

Thefigureshownbelowisaplotofthereciprocaloftheinitialrateofuptakeof Dglucose(1/v(Dglucose))bymousetumorcellsversusthereciprocalof extracellularDglucoseconcentration(1/[Dglucose])whenmeasuredinthe absence(Dglucosealone)orinthepresenceof10mMDgalactose(Dglucose+ 10mMDgalactose).

OnthebasisoftheseresultswhatisthelikelymechanismofDglucoseuptakeby thissystem?Assumethat:
v(Dglucose)=Vmax[Dglucose] Km+[Dglucose] where,v(Dglucose)=initialrateofDglucoseuptake Vmax=maximalrateofDglucoseuptake Km=concentrationofDglucosewhenv=Vmax/2

InwhatwaydoesDgalactoseappeartoinfluencethekineticsofDglucose uptake?Explainyourreasoning.
(5 pts) The addition of 10 mM D-galactose to the uptake medium does not change the magnitude of the 1/v(D-glucose) intercept. Given that the kinetics of D-glucose uptake approximate Michaelis-Menten kinetics (yield a straight line relationship when 1/v(Dglucose) is plotted against 1/[D-glucose], this implies that D-galactose does not decrease or increase the maximal capacity of the transporter. When 1/[D-glucose] = 0, 1/v(D-glucose) = 1/Vmax; v(D-glucose) = Vmax. However, D-galactose does increase Km. When 1/v(D-glucose) = 0, Km/Vmax[D-glucose] = -1/Vmax and 1/[D-glucose] = -1/Km. In words, the addition of 10 mM D-galactose to the uptake medium results in a less negative value for -1/Km, meaning that the absolute value of Km is increased. Dgalactose therefore appears to be a simple competitive inhibitor of D-glucose transport. That the transport of D-glucose is both saturable and stereospecific in so far as D-galactose competes with D-glucose for uptake, implies that the transport observed is carrier-mediated.

BIOL121 Exam1 10/02/09

NAME:

IfinthecourseoftheseexperimentsyoudeterminethattheinitialrateofD glucoseuptakeissome50,000timesfasterthattherateofuptakebysimple passivediffusionbutnoteffectedbyiongradientsacrosstheplasmamembrane, forinstancethoseforNa+and/orK+,orbyintracellularATPlevelswhatisthe likelymechanismofDglucoseuptakeinthissystem.Whatothermechanismsfor Dglucoseuptakemightbeexcludedonthebasisofthesefindings?Explainyour reasoning.


(5 pts) It is improbable that the transport observed is subject to primary energization because it is not influenced by intracellular ATP levels. Likewise, since neither Na+ nor K+ influence the velocity of D-glucose uptake, it is unlikely to be by a mechanism involving Na+ and/or K+ symport or antiport, i.e. it is unlikely to be secondarily energized by monovalent cations. On this basis and its high velocity by comparison with simple diffusion, in combination with its stereospecificity and saturability, the transport measured appears to be by facilitated diffusion.

Wouldthetransportpathwayyouhaveidentifiedbecapableofmediatingthe accumulationofDglucosebymousetumorcells?Explainyourreasoning.
(5 pts) No. Facilitated diffusion simply serves to accelerate equilibrative transport. It cannot establish a concentration gradient because it is freely reversible and not susceptible to primary or secondary energization by factors that impose directionality on the transporter.

Giventhatmanytumorcellsexhibitincreasedglycolyticactivity,whatfactoror factorsensuresustainedglucoseuptakebythisDglucosetransporter?Explain yourreasoning.


(5 pts) A prerequisite for the glycolytic metabolism of D-glucose is its rapid phosphorylation by ATP to give glucose-6-phosphate (G6P). What this means, especially if glycolytic activity is high in tumor cells, is that the glucose that enters the cell is metabolized so maintaining intracellular glucose at a low concentration relative to its concentration in the bathing medium. The transporter identified is thereby capable of sustained glucose uptake because glucose metabolism via the glycolytic pathway ensures that the glucose concentration gradient is inwardly directed. If glucose was not converted to something else soon after its entry into the cell its intracellular concentration would steadily rise with time to a point at which intracellular concentration = extracellular concentration and net flux is zero.

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BIOL121 Exam1 10/02/09

NAME:

14) BondswhosehydrolysisproceedswithlargenegativevaluesofG(customarily morenegativethan6kcal/mol;25kJ/mol)areoftenreferredtoas"highenergy bonds".Itisimportanttonote,however,thatnoneofthebondsofATPhave especiallyunusualproperties.Theterm"highenergybond"(sometimes denotedasADP~P)whenappliedtothiscompoundcanthereforebemisleading. ExplainandcommentonATPsstatusnotsomuchasaveryhighenergy phosphatebutmoreasanintermediateenergyphosphate.Whatbearing doesthishaveonourunderstandingoftheprimingreactionsintheenergy investmentphaseofglycolysisandthesubstratelevelphosphorylationsinthe energygenerationphase?
(12 pts)

The phosphate bonds of ATP are not particularly strong bonds. Instead, the "high energy" character of ATP derives, in part, from its charge distribution. In the ATP molecule all three phosphate bonds are negatively charged. These "like charges" are crowded together, and their repulsion contributes to the instability of this region of the ATP molecule. ADP, on the other hand, is much more stable. In the physiological pH range, ATP has three to four negative charges whose mutual electrostatic repulsion is partially relieved by its hydrolysis to ADP. The crucial role of ATP perhaps has less to do with its status as a high energy phosphate but more to do with its status as an intermediate energy phosphate. An important feature of the ATP-ADP system is that it is a primary connecting link between high- and low-energy phosphate compounds. ATP is of intermediate phosphate transferring potential. ADP can accept a phosphate group from a compound with a higher free energy of hydrolysis, for instance 1,3bisphosphoglycerate (1,3-bisPG) and phosphoenolpyruvate (PEP) in the energygeneration phase of glycolysis by substrate-level phosphorylation, and the ATP so formed can then donate it to an acceptor molecule to yield a phosphate compound lower on the scale, for instance the phosphorylated sugars glucose-6-phosphate (G6P) and fructose-1,6-bisphosphate (F16bP) generated in the priming reactions of the energy-investment phase of glycolysis. For this to happen (to be thermodynamically spontaneous) it is imperative that the free energy of hydrolysis of ATP is less than those of 1,3-bisPG and PEP and higher than that of G6P and F16bP.

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