8/9/2019 Biochem Exam 3_2013

1/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

+

Biochemistry Exam 3

Biology 020.305

November 13, 2013Instructions for examination:1. BEFORE YOU BEGIN, clearly print your name and JHED ID ON THE TALLY PAGE. Failure to

write your JHED ID will cost 2 points. Please also write your name on the front of every page in casethe pages get separated.

2. There are a total of 14 pages including: 1 cover page, 10 question pages 2-11, 1 tally page 12, 1metabolism pathway diagram page 13 and 1 scratch page 14. If there are any pages missing, obtain anew exam. No excuses will be accepted for missing pages.

3. Write answers clearly and concisely in the space provided. Anything written on the back of theexam is not graded. Extra space for doodling is found on the last page of the exam. If you desirecredit for anything that is NOT in the answer space provided, you must clearly indicate where we

should look for that information.

4. For multiple choice questions with multiple possible correct answers (i.e. Circle all that apply), you

will get full credit for selecting all correct answers only. Points may be deducted for selecting incorrectanswers with a minimum score of 0.

5. Exams not written in blue or black permanent ink or containing white-out will not be regraded.

6. Calculators are not necessary and their use is not allowed.

7. This exam is closed note and closed book. Books, notes, phones, computers etc., are to be

placed out of sight before the beginning of the exam and are not to be referred to during the exam.Talking or looking at other exams during the test is prohibited.

8. If you have any questions, raise your hand and a TA or instructor will come to you. If there isanything about the exam room that is negatively affecting your ability to focus on the exam, it is your

responsibility to notify a TA or instructor.

9. You will be allowed 65 minutes to take this exam. Once time is called you must stop writing. Any

writing after time is called will result in the loss of points and may be reported as an ethics violation.

10. When you are done with the exam, please show your ID to a proctor who will accept your exam.Then sign out by marking your test number on the roster.

11. Ethics Pledge: Please make the following commitment by signing and dating underneath thefollowing pledge: I agree to complete this exam without unauthorized assistance from any person,

materials, or device. I will not alter my answers in any way after receipt of the graded exam. Your

signature here indicates that you have read and understood items 1 -10. Tests not signed may not be

handed back or regraded.___________________________________

Good Luck!

8/9/2019 Biochem Exam 3_2013

2/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

"

1. 19 points total. Fermentation and Respiration. Pyruvate produced from glycolysis can be used

for fermentation or for cellular respiration. When oxygen is supplied, pyruvate produced from

glycolysis is converted to acetyl coenzyme A for cellular respiration.

A. Pyruvate dehydrogenase, which converts pyruvate to acetyl coenzyme A, is tightly regulated at

multiple levels. If the ratio of NADH to NAD+ in the mitochondria is elevated (more NADH), pyruvate

dehydrogenase activity will: INCREASE or DECREASE (circle one). 1

B. When pyruvate produced from glycolysis is used for cellular respiration, how many molecules of

ATP will be produced per each pyruvate consumed? Assume that mitochondrial NADH and FADH2

are equivalent to 2.5 and 1.5 molecules of ATP, respectively. _______ 2

Answer: 4 NADH + 1 FADH2 + 1 ATP = 10 + 1.5 +1 = 12.5 ATP.

1pt for 10

When oxygen is absent, pyruvate produced from glycolysis is used for fermentation to lactate.

C. What is the cofactor required for the fermentation of pyruvate to lactate? ____________ NADH 2

D.How many molecules of ATP will be produced by fermentation of pyruvate to produce lactate?

Assume that mitochondrial NADH and FADH2 are equivalent to 2.5 and 1.5 molecules of ATP,

respectively. _____0 (2)

E. If fermentation is occurring in muscle cells during exercise, lactate produced from glycolysis needs

to be transported out of cells so that glycolysis can continue. The transporter of lactate requires one

additional substrate in addition to lactate. 2

i. What is the additional substrate? _________ proton

ii. In which direction is this additional substrate transported?

Circle one: Same direction as lactate Opposite direction from lactate

F. The accumulation of lactate secreted into the blood can be lethal if it is not removed. Liver is the

major organ that removes lactate from the blood. Name two possible metabolic pathways that could

use lactate in the liver. Explain how lactate enters the pathway naming key intermediates. 4

Pathway 1: gluconeogenesis, TCA, fatty acid synthesis, pentose phosphate, cholesterol synthesis,alanine synthesis

Explain: via conversion to pyruvate and or acetyl CoA, OAA ok for gluconeogenesis

Pathway 2:

Explain:

8/9/2019 Biochem Exam 3_2013

3/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

$

1. continued

G. The production of lactate by muscle cells and uptake of lactate by liver cells is part of the Cori cycle.

In the Cori cycle, how many molecules of ATP are consumed per cycle? ___4 ATP 2

H. When cells are depleted of oxygen and, thus, electron transfer chain is not functional, cells do not

normally convert pyruvate to acetyl coenzyme A. If cells did convert pyruvate to acetyl coenzyme A

and if the acetyl coenzyme A is used for citric acid cycle, how many molecules of ATP will be

produced from a molecule of pyruvate consumed? _1 ATP____2

I. Conversion of pyruvate to acetyl CoA for use in the citric acid cycle is energetically favorable even

in the absence of oxygen. However, normal muscle cells do not use respiration in the absence of

oxygen. Respiration is likely inhibited due to the accumulation of NADH in mitochondria in the

absence of oxygen. How can NADH accumulation in mitochondria inhibit respiration? 2

Answer: NADH inhibits TCA cycle, specifically pyruvate dehydrogenase, isocitrate dehydrogenase,and succinyl CoA synthase.

2. 22 points total. Cellular compartmentalization of metabolic pathways and Gluconeogenesis.

A. Yeast cells are eukaryotes and they normally have mitochondria. However, some yeast cells called

rhoohave almost no mitochondrial functions but are still viable. Please select all metabolic processes

rhoocannot do. Select all that apply. 3

i. glycolysis

ii.citric acid cycle

iii.oxidative phosphorylation

iv. fatty acid oxidation

v.

cholesterol synthesis

vi.pentose phosphate pathway

vii.

nucleotide biosynthesis

viii.

fatty acid synthesis

In comparison to some of the metabolic pathways above, gluconeogenesis requires multiple

subcellular organelles/compartments.

B. The first step of gluconeogenesis is the conversion ofpyruvate to oxaloacetate, where ATP is

consumed. This step occurs in: Select one answer. 2

i. mitochondrial inner membraneii. cytoplasm

iii. endoplasmic reticulum

iv. plasma membrane

v. mitochondrial matrix

vi. mitochondrial outer membrane

vii. mitochondrial intermembrane space

8/9/2019 Biochem Exam 3_2013

4/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

4

2 continued

The second step of gluconeogenesis is the conversion of oxaloacetate to phosphoenolpyruvate,

where GTP is consumed. For this reaction, oxaloacetate produced in the mitochondria needs to be

transported to the cytosol.

C. Fill in the blanks. Oxaloacetate is first converted to ____________ using ____________ as a co-

substrate. The product of this reaction is transported to the cytosol, then converted back to

oxaloacetate using ___________________ as a co-substrate. 3

Answer: malate, NADH or NADH + H+, NAD+.

D. How many molecules of ATP or GTP should be hydrolyzed to make one molecule of glucose from

pyruvate? ______Answer: 6 ATP or GTP. 2

1 point for 3 ATP

The conversion of fructose-1,6-bisphosphate to fructose-6-phosphateis not simply the reverse of

glycolysis and requires the enzyme (F6BPase). F6BPase is inhibited by fructose-2,6-bisphosphate(F2,6BP) and activated by citrate.

E. Is FBPase stimulated or suppressed when ATP citrate lyase becomes hyperactive?

Circle One: Stimulated or suppressed 1

The molecule shown below (PFKFB) catalyzes both the conversion of F6BP to F2,6BP (PFK2) and

F2,6BP to F6BP (FBPase2). The figure shows the reciprocal regulation of the enzyme by

phosphorylation.

F. When liver cells are stimulated with glucagon:

Circle the correct words of each underlined pair to correctly complete each sentence. 5

i. (Circle one: PKAor phosphoprotein phosphatase) will be (Circle one: activatedor inhibited).

ii. PFKB will be (Circle one: phosphorylatedor dephosphorylated).

iii. F26BP levels will (Circle one: increase or decrease).

iv. Blood glucose levels will (Circle one: increaseor decrease).

8/9/2019 Biochem Exam 3_2013

5/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

%

2 continued

The last step of gluconeogenesis is the hydrolysis of glucose-6-phosphate to

glucose and inorganic phosphate by glucose-6-phosphatase. Breakdown of

glycogen generates glucose-6-phosphate which can also be a substrate for

Glucose-6-phosphatase.

G. In which subcellular location does this step occur? Select one answer. 2

i. mitochondria inner membrane

ii. cytoplasm

iii. endoplasmic reticulum

iv. plasma membrane

v. mitochondrial matrix

H. How is this gluconeogenic reaction differentthan the conversion of glucose to glucose-6-

phosphate during glycolysis? Select all that apply. 2

i. the gluconeogenic reaction occurs in different cellular location

ii. the gluconeogenic reaction generates ATP instead of using ATPiii. the gluconeogenic reaction occurs regardless of the concentration of substrates

iv. the glucogenogenic reaction is thermodynamically unfavorable

I. The reaction is regulated by transporting the substrate and products in and out of the subcellular

organelle. If the transport of inorganic phosphate out of the subcellular organelle were inhibited, which

of the following would be true? Select one answer. 2 1 pt for selecting 2/3 answers

i. the reaction will be inhibited

ii. gluconeogenesis will be inhibited

iii. glycogen breakdown will be decreased

iv. all of the above

v. none of the above

3. 10 points total. Pentose phosphate pathway and nucleotide biosynthesis. In the pentose

phosphate pathway, glucose-6-phosphate is converted to ribose-5-phosphate. Glucose-6-phosphateis an intermediate of metabolic processes.

A. Which metabolic processes produce glucose-6-phosphate? Select all correct ones. 2

i. glycolysisii. gluconeogenesisiii. fatty acid synthesis

iv. fatty acid oxidation

v. citric acid cycle

B. How many molecules of ATP are generated by the conversion of 4 glucose-6-phosphatemolecules to pyruvateduring glycolysis (ignore fermentation/respiration)? _____ 2Answer: straight through glycolysis 3 ATP each x 4 = 12 ATP; 1 point for 3, 1 point for 8Instead, the 4 glucose-6-phosphate molecules can enter the pentose phosphate pathway to produce

2 fructose-6-phosphate molecules, 1 ribose-phosphate, and 1 glyceraldehyde-3-phosphate.

C. In this case, how many molecules of ATP will be generated from the combination of pentosephosphate pathway and glycolysis (ignore fermentation/respiration)? ____ 2

through pentose phosphate: 2 F6P x 3 ATP + 1 GAP x 2 ATP = 8 ATP.

8/9/2019 Biochem Exam 3_2013

6/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

5

3 continued

D. How many molecules of NADP+are reduced from the 4 molecules of glucose-6-phosphate? _____

2 Answer: 2 NAD PH per glucose-6-phosphate x 4 glucose -6-phosphate = 8 NADPH,E. If all of the NADPH generated from 4 molecules of glucose-6-

phosphate (answer above) are used for the synthesis of a fatty

acid called hexanoic acid (C5H11CO2H), how many molecules of

the fatty acid can be produced? _______ 2

Answer: for each synthesis of hexanoic acid, 2 cycles of fatty acid synthesis is required. Each

cycle consumes 2 NADPH. Thus 4 NADPH are consumed. Therefore, the NADPH produced from C is

sufficient to make 2 hexanoic acids

4. 16 points total. Fatty acid metabolism.

A family of molecules found in coffee called coffee polyphenols

(CPP) have been shown to affect mice that have been fed a high-fat

diet. Specifically, mice on the high-fat diet who were given CPPshowed lower triglyceride levels compared to mice on the same

high-fat diet with no CPP.

A. Triglyceride obtained from food that enters the bloodstream can

be used for immediate energy or storage. 2

i. Name one type of cell that will use triglcyerides immediately for

energy. ____ muscle

ii. Name one type of cell that will store triglcyerides. ____ liver, adiopcyte

Researchers decided to measure both fatty acid synthesis and !-oxidation to determine the

mechanism of CPP function.

B. Both fatty acid synthesis and breakdown (beta-oxidation) require shuttles for critical molecules to

cross the inner mitochondrial membrane at the beginning of the processes. Select one of these

shuttles and answer the following questions. 3

Select one: Carnitine shuttle Citrate shuttle

i. Which metabolic Pathway requires this shuttle?

Circle one: Fatty acid synthesis or Beta-oxidation

ii. Name the key molecule transported that will continue in the pathway. _____________

iii. Is the molecule transported into or out of the mitochondrial matrix?

Circle one: Into matrix or Out of matrix

Carnitine shuttle: beta oxidation, acyl-CoA, into matrix

Citrate shuttle: fatty acid synthesis, acetyl-CoA, out of matrix

8/9/2019 Biochem Exam 3_2013

7/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

6

4 continued

C. This graph shows that mice on a high fat diet have lower levels of

Acetyl-CoA carboxylase (ACC) activity when treated with CPP. What

conclusions about the effects of CPP are consistent with this

observation? Select all that apply. 2

i. suppresses fatty acid synthesis

ii. increases malonyl-CoA levels

iii. decreases acetyl-CoA levels

iv. suppresses beta oxidation

v. promotes cancer cell survival

D.The following molecules are regulators of ACC activity that might be affected by CPP. Which

molecules normally inhibitACC activity? Select all that apply. 3

i.AMP-activated kinase (AMPK)

ii. citrate

iii. long chain fatty acidsiv. insulin

v. glucagon

E.The graph shows the oxygen consumption of control

mice on a normal diet (Cont), mice on a high fat diet (HF),

and mice on a high fat diet supplemented with CPP (HF +

1.0% CPP). What is the best conclusion can you make

from this data? Select one. 4

i. a high fat diet increases ATP synthesis

ii. a high fat diet decreases fatty acid !-oxidation

iii. CPP stimulates aerobic fermentation

iv. CPP increases fatty acid !-oxidation

v. CPP decreases ATP synthesis

Explain your reasoning:

More O2 is consumed, so the ETC is active. This means the TCA cycle is active and the likely source

of acetylCoA is from fatty acid breakdown.

It cannot be the high fat diet alone because the HF mice use the same amount of oxygen as the

control mice.

F. The data showed that, in addition to fatty acid synthesis, cholesterol synthesis is affected. Fattyacids synthesis and cholesterol synthesis both: Select all that apply. 2

i. use NADH.

ii. can be synthesized from acetyl CoA.

iii. require HMG-CoA reductase.

iv. require Acyl carrier protein (ACP).

v. can be regulated by SREBP.

8/9/2019 Biochem Exam 3_2013

8/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

7

5. 9 points total. Metabolism and Cancer

Metabolic pathways of cancer cells are often observed to be different than normal cells.

A. The Warburg effect is characterized by an increase in: (Select all that apply). 2

i. fatty acid synthesis

ii. glycolysis

iii. oxidative phosphorylation

iv. gluconeogenesis

v. aerobic fermentation

vi. anaerobic fermentation

vii. nucleotide synthesis

The citric acid cycle is also altered in cancer cells. In some cancer cells, Isocitrate dehydrogenase

(IDH) converts isocitrate to "-ketoglutarate and another mutant isocitrate dehydrogenase additionally

converts reduces "-ketoglutarate to 2-hydroxyglutarate using NADH.

B. If every "-ketoglutarate generated by TCA is immediately converted to 2-hydroxyglutarate (andexcess oxaloacetate is available), how many molecules of ATP will be produced from each molecule

of acetyl coenzyme A in this particular case?______0 ATP. 2

Another alteration of the TCA cycle observed in cancer cells is the conversion of isocitrate to

glyoxylate. Glyoxylate can react with acetyl-CoA to form malate, bypassing several steps of the TCA

cycle.

C. Compared to normal cells, cancer cells using the glyoxylate pathway will have which of the

following? Select all that apply. 1

i. higher oxaloacetate levels

ii. higher succinyl-CoA levels

iii. lower isocitrate dehydrogenase (IDH) levels

iv. lower ratio of citrate to malate

v. higher ratio of isocitrate to "-ketogluterate

D. Name one advantage that bypassing specific steps of the TCA cycle potentially provides to cancer

cells. 2

Advantage: able to use pyruvate without being regulated by IDH and alpha-ketogluterate

dehydrogenase

E. How might the glyoxylate pathway contribute to increased glycolysis in cancer cells? 2

Possible answers include glyoxylate pathway provides malate and oxaloacetate that can be used to

consume cytoplasmic NADH, it can be used to convert pyruvate to other biological materials!etc

8/9/2019 Biochem Exam 3_2013

9/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

8

6. 12 points total.Signaling and Diabetes

The insulin signaling pathway is diagrammed in the image above. To summarize, insulin binding to

the receptor leads to activation of the kinase Akt. Active Akt affects the cell in several ways including

causing an increase in the amount of the glucose transporter GLUT4 on the cell surface.

A. Which of the following is true about insulin signaling? Select all that apply.2

i. Insulin is secreted by liver cells

ii. Insulin is secreted by pancreas cells

iii. Insulin signaling leads to increased uptake of glucose from the bloodiv. When insulin is secreted, blood glucagon concentration is elevated.

B. Which pathways are stimulated by insulin signaling? Select all that apply.3

i. glycolysis

ii. gluconeogenesis

iii. breakdown of glycogen

iv. synthesis of glycogen

v. beta-oxidation

vi. fatty acid synthesis

Type II diabetes is often called insulin-resistant diabetes, meaning that insulin is properly secreted,

but other cells do not respond to insulin properly.

C. Which of the following scenarios would likely cause an insulin-resistant phenotype? Select all that

apply. 2

i. An insulin receptor that constantly phosphorylates its substrates

ii. A version of Phopsphoinositide 3 kinase that does not localize to the cell plasma membrane

iii. A version of PDK1 that cannot bind PIP3

iv. A version of Atk that is constantly phosphorylated

v. A cell membrane with excessively high levels of PIP3 in the absence of PI3K activity

D. Select one of the answers you chose in part B above and briefly (2 sentences or fewer) describe

how this would lead to the type II diabetes phenotype.

3ii:If PI3K were not at the membrane, it would be slow to find its substrate (which is part of the

membrane), PIP3 would not be made efficiently, PDK would not activate Akt and Akt would not cause

GLUT4 to be in the membrane, so blood glucose would not be imported leaving blood glucose levels

high.

iii.If PDK1 does not bind PIP3, it will not be activated, will not activate Akt, and Akt would not cause

GLUT4 to be in the membrane, so blood glucose would not be imported leaving blood glucose levels

high.

8/9/2019 Biochem Exam 3_2013

10/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

+!

6 continued

E. Individuals with type II diabetes are likely to also have defects in glucagon signaling. 2

Is this statement true or false? Circle one: True False

Explain your reasoning:

If False: Glucagon signals through a completely different mechanism including a different receptor

(beta adrenergic) and signaling molecules including cAMP.

If TRUE: an explanation might be that these individuals have overactive glucagon signaling that

causes liver cells to undergo gluconeogenesis constitutively, thus moving glucose into the blood,

elevating glucose levels)

7. 12 points. ETC and ATP Synthase

A. On the diagram of the electron transport chain above, what molecule is represented by D?

______ 1 Cytochrome C

The table lists 3 inhibitors of the electron transport chain.

!"#$%$&'( *&+, $"#$%$&+-

!"#"$%& (%#)&"* +

,-$.%*/0 (%#)&"* ++

102/#3(/0 1 4 (3(&"

B. Compare the effects of these three drugs on the following: 6

Delivery of electrons from NADH and FADH2:

Demerol inhibits delivery of electrons from NADH

Carboxin inhibits delivery of electrons from FADH2

Antimycin A inhibits both. Credit was given if the answer states that ActA has no direct effect on

electrons being delivered to Q !QH2

Proton Gradient:

Demerol suppress all proton gradient from NADH but not from FADH2

Carboxin inhibits all proton pumping from FADH2 (which includes the protons normally pumped from

complexes III and IV) but not from NADH

Antimycin A inhibits proton pumping from both NADH and FADH2 (partial credit if one notes antimycin

A still produces 4H+ per NADH)

ATP synthesis:

All three reduce ATP synthesis. Actimycin A results in the least protons pumped, so reduces ATP

synthesis the most. Demerol causes a moderate decrease in ATP synthesis, and carboxin has the

smallest effect. These effects are directly related to the number of protons that can be pumped in the

presence of each drug.

8/9/2019 Biochem Exam 3_2013

11/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

++

7 continued.

C. In the c subunit of F1 F0 ATP synthase, there is a conserved aspartate residue that binds to H+. If

the aspartate is mutated to a non-natural amino acid of equal size with a primary amine side chain,

what will happen to the rotation of the F0 subunit? 1

i. still rotate

ii. stop rotating

iii. rotate in the other direction

D. Electrons from NADH produced in the cytosol are transported to the mitochondria indirectly by the

malate-aspartate shuttle or the glycerol-3-phosphate shuttle. 2

i. Why might the malate-asparate shuttle be preferred? (One sentence limit). More ATP

more efficient, NADH conserved

ii. Why is this shuttle not used by all cells? (One sentence limit). Reversible- it only works

when cytosolic NADH/NAD+ ratio is higher than mitochondrial NADH/NAD+ ratio.

Typical F1 F0 ATP synthase contains 12 c subunits and mitochondrial NADH adds 10 protons to the

gradient. Imagine that there is an organism that has 3 c subunits instead of 12.

E. In this organism, how many ATP can be produced from NADH oxidation? Please take into

account that the transport of ADP from cytoplasm to mitochondrial matrix consumes 1 H+.

______ 2

Answer: 3 H+ equals 3 ATP in this system. Thus 1 H+ = 1 ATP. And because of ADP transport, 1

more H+ is needed. Total 2 H+ per ATP. NADH=10 protons=5 ATP

REMEMBER TO WRITE YOUR JHED ID ON THE FOLLOWING PAGE

8/9/2019 Biochem Exam 3_2013

12/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

+"

Tally Page-WRITE YOUR JHED ID ON THIS PAGE

page 2 ______________________ of 13

page 3 ______________________ of 11

page 4 ______________________ of 11

page 5 ______________________ of 12

page 6 ______________________ of 9

page 7 ______________________ of 11

page 8 ______________________ of 9

page 9 _____________________ of 10

page 10 _____________________ of 9

page 11 _____________________ of 5

-2 if no jhed ID ___________________

total _______________________ of 100

8/9/2019 Biochem Exam 3_2013

13/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

+$

8/9/2019 Biochem Exam 3_2013

14/14

!"!#$!% '()* $ "!+$ ,)*- ............................. /0-1 23............................

+4

SCRATCH PAGE