7.05 Spring 2004

April 16, 2004

Recitation #8 Contact Information TA: Victor Sai E-mail: [email protected] Unit 3 Schedule Recitation/Exam Recitation #8 Exam 3 Review Exam 3

Date Friday, April 16 Monday, April 19, 7pm, 10-250 Wednesday, April 21, Walker

Recitation: Office Hours:

Friday, 3-4pm, 2-132 Friday, 4-5pm, 2-132

Topics Sugar Metabolism, Pathway Game Lectures 19-26 Lectures 19-26

Recitation Overview Topic 1. Sugar Metabolism 2. Playing the Pathway Game

Problems 1, [5] 2-4, [6], Quiz

Problems 1. (1998 Exam 2 Question 3, 30 points) Give the reaction sequence that would allow the overall enzymatic transformation indicated by the following equation. Note that both (glucose)n and (glucose)n+5 refer to glycogen. Sucrose + lactose + mannose + (glucose)n

(glucose)n + 5

In presenting your answer, assume the presence of adequate amounts of nucleoside triphosphates. You are not required to use structural formulas, but be sure to name the reactant(s) and product(s) for all of the proposed enzymatic reactions in the sequence. Use only those enzymes that have been discussed in class. Indicated how many ATP’s are needed to allow the overall transformation to occur.

Recitation 8

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7.05 Spring 2004

April 16, 2004

ATP Sucrose

ADP

Fructose

Fructose-1-P

Glucose ATP Glyceraldehyde

Dihydroxyacetone-P

ADP

ATP

Glucose-6-P

ADP Glyceraldehyde-3-P Lactose

ATP

ADP

Glucose-6-P

Glucose Galactose ATP

Fructose-1,6-di P

ADP Galactose-1-P UDP-Glucose

Pi Fructose-6-P epimerase

UDP-Galactose

Glucose-6-P

Glucose-1-P

ATP

ADP

Mannose

4 Glucose-6-P

Mannose-6-P

P-Glucomutase 5 UTP 5 PP i

5 Glucose-1-P

Isomerase

Fructose-6-P

Isomerase

Glucose-6-P

4 Glucose-1-P (Glucose)n (Glucose)n+5 5 UDP-Glucose

5 UDP 5 ATP 5 ADP 5 UTP

11 ATP needed

Recitation 8

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7.05 Spring 2004

April 16, 2004

2. (1999 Exam 2 Question 4, 25 points) a) In the presence of O2 and arsenite (not arsenate!), liver tissue was observed to metabolize 0.002M fumarate and 0.002M acetyl CoA completely. What would the products be and how much of each product would be formed? Please give the enzymatic reactions to account for your answer (no formulas are necessary). How much ATP could be produced during this transformation? Hint: arsenite reacts with dimercapto compounds.

2 H2O

+ 2 NAD+ 2 NADH + 2H

2 Fumarate

2 Malate

2 H2O 2 CoA

2 Citrate

2 Oxaloacetate 2 Acetyl CoA

2 H2O

2 H2O

2 NADH + 2H+ 2 NAD+

2 cis-Aconitate

2 Isocitrate

2 α-ketoglutarate 2 CO2

α-ketoglutarate cannot be converted to Succinyl CoA because arsenite reacts with the dimercapto form of Lipoic acid, which is produced during the action of α-ketoglutarate oxidase. Thus α-ketoglutarate would be the end product along with 2 CO2. Since 4 NADH are produced, 4 x 3 = 12 ATP could be generated.

b) What would the products be if arsenite and acetyl CoA are deleted from the incubation mix and 0.4M malonate is added? Please show how your answer was obtained (again, no formulas are necessary). How much ATP could be generated?

2 H2O

+ 2 NAD+ 2 NADH + 2H

2 Fumarate

2 Malate

ADP + Pi ATP

2 Oxaloacetate (1)

1 Pyruvate

(1) HCO3

H2O

cis-Aconitate

CoA

NAD+

CO2

NADH

1 Acetyl CoA

Citrate

H2O NAD+

-

CoA

H2O

NADH + H+

NAD+

NADH + H+

α-ketoglutarate

Isocitrate CO2

GDP + Pi

Succinyl CoA CoA

GTP

Succinate

CO2

Thus one mole of Succinate would be produced per 2 moles of Fumarate used. Four moles of CO2 would also be made along with 17 ATP.

Recitation 8

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7.05 Spring 2004

April 16, 2004

3. (1998 Exam 2 Question 4, 25 points) A microorganism is known to be able to carry out the following overall enzymatic transformation under aerobic conditions and with a relatively high cellular ratio of ATP and ADP. 3 pyruvate

3 CO2 + Citrate

Give the individual enzymatic reactions (no structural formulas needed) that account for this transformation. Use only enzymatic reactions that have been discussed in class. Please note that CO2 is produced but no CO2 is utilized. How many ATP’s could be produced in this process?

3 Pyruvate 3 CoA

3 NAD+

3 CO2

3 NADH

3 Acetyl CoA (1)

Oxaloacetate +

NADH + H

(1)

(1) H O 2

CoA

Citrate

+

NAD

Malate

H2O

CoA

cis-Aconitate H2O

H2O

Glyoxylate Isocitrate Succinate

+ NAD+ NADH + H

H2O

Fumarate

FAD

Malate

FADH2

Oxaloacetate H2O CoA

During this process 5 NADH are formed and 1 FADH2 are produced, which could lead to the production of 17 ATP

Recitation 8

Citrate

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7.05 Spring 2004

April 16, 2004

4. (1996 Exam 2 Question 5, 25 points) Assume that a bacterial species that is missing the enzyme isocitrate dehydrogenase can, under aerobic conditions, convert 0.004M pyruvate to 0.006M CO2 and 0.001M glucose-6-P in the presence of adequate amounts of inorganic phosphate and ADP. Give the set of enzymatic reactions that account for this transformation and indicate how much net ATP could be generated from the overall transformation. Please note that CO2 is produced, but no CO2 is used (i.e., no carboxylation reactions occur). The only compounds that are used in substrate quantities are pyruvate, ADP, and Pi. Any other compounds that may be needed must be regenerated in subsequent reactions. No structural formulas are required but be sure to indicate which, if any, coenzymes may be needed for individual enzymatic reactions.

Recitation 8

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7.05 Spring 2004

April 16, 2004

4 Pyruvate 4 CoA

4 NAD+

4 CO2

4 NADH

4 Acetyl CoA

(2) 2 H2O

(2)

2 Oxaloacetate 2 NADH + 2 H+

2 CoA

2 Citrate

+

2 NAD

2 Malate

2 H2O

2 CoA

2 cis-Aconitate 2 H2O

2 H2O

2 Glyoxylate 2 Isocitrate 2 Succinate

2 H2O

2 Fumarate

2 FAD

+ 2 NAD+ 2 NADH + 2 H

2 Oxaloacetate

2 Malate

2 FADH2

2 GTP 2 GDP

2 3-P-Glycerate

2 PEP

2 2-P-Glycerate

2 ATP

2 CO2

2 H2O

2 ADP

+ 2 NADH + 2 H+ 2 NAD

2 1,3-di P-Glycerate

2 Glyceraldehyde-3-P (1)

1 Fructose-1,6-di P

(1)

1 Dihydroxyacetone-P

H2O Pi

1 Fructose-6-P

1 Glucose-6-P

6 net NADH = 18 ATP 2 net FADH2 = 4 ATP -4 net ATP (2 ATPs + 2 GTPs used) Net Yield = 18 ATP

Recitation 8

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7.05 Spring 2004

April 16, 2004

Practice Problems 5. (1996 Exam 2 Question 3, 25 points) Give a set of proposed enzymatic reactions that would allow the disaccharide shown below to be converted to 2 glucose units of glycogen. Assume the presence of any other compounds that may be needed for this overall conversion. No need to use structural formulas unless you wish to do so. What type of enzyme would be needed to begin the reaction sequence (i.e. to convert the disaccharide to 2 monosaccharides)? CH 2OH OH

O OH

O

CH2

O

CH2OH

HO

OH

HO OH

Recitation 8

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7.05 Spring 2004

April 16, 2004

The given dissacharide can be cleaved into galactose and fructose by β-galactosidase ATP Galactose ATP

Fructose

ADP Galactose-1-P UDP-Glucose

ADP Fructose-1-P

Dihydroxyacetone-P

Glyceraldehyde ATP

epimerase

UDP-Galactose Glucose-1-P UTP

ADP Glyceraldehyde-3-P

PPi UDP-Glucose (Glucose)n UDP (Glucose)n+1

Fructose-1,6-di P Pi Fructose-6-P

Glucose-6-P

Glucose-1-P UTP PPi UDP-Glucose (Glucose)n UDP (Glucose)n+1

Recitation 8

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7.05 Spring 2004

April 16, 2004

6. (1999 Exam 2 Question 5, 20 points) Under aerobic conditions a bacterial species was shown to metabolize completely 0.002M pyruvate in the presence of 0.1M malonate and under conditions in which isocitrate dehydrogenase is inoperative. What would the products be and how much of each would be formed? How much ATP could be formed? Please show how your answer was obtained (no formulas are necessary).

2 Pyruvate 2 CoA

2 NAD+

2 CO2

2 NADH

2 Acetyl CoA (1)

Oxaloacetate NADH + H+

(1) H O 2

CoA

Citrate

NAD+

Malate CoA H2O

H2O

cis-Aconitate

3 NADH are formed; therefore 3 x 3 = 9 ATP could be generated

H2O

Glyoxylate Isocitrate Succinate

Recitation 8

Page 9 of 9

Metabolism of Sugars and Their Entry to Glycolysis

C.Allen & S.Chen 7.05 Spring 2001

This handout summarizes the metabolic breakdown of lactose, galactose, sucrose and mannose, and their entry to the glycolysis. Boxed compounds are intermediates of glycolysis. Lactose Metabolism: including the breakdown of Galactose

OH

OH

OH

O

O

OH

1

O 4

OH

Glucose

OH

OH

ATP

β-galactosidase

HO

HO

galactose-1-phosphate uridylyl transferase

O

NAD+ NADH+H+

OH O

OH HO

OH

OH

O

O

P O

P O Uridine

O-

O-

UDP-Glucose

O

O

OP

OH HO

O-UDP

enzyme bound intermediate

NADH+H+

phosphoglucomutase

OH

NAD+

Glucose-1phosphate

OH

UDP-galactose-4-epimerase HO

Galactose-1phosphate

OP

Galactose OH

OH

O

galactokinase

HO

Lactose

OH OH

OH

β-D-galactopyranosyl-(1->4)(α or β)-D-glucopyranose

O

ADP

OH

OH

HO

OH

O

OH

O OH O

HO

O

O

P O

P O Uridine

O-

O-

UDP-Galactose

OP O

Glucose-6phosphate

OH OH

OH HO

C. Allen & S. Chen 7.05 Spring 2001

Sucrose Metabolism OH HO

O

O

OH

Glucose

HO

OH O

HO

OH

O

ATP

ADP

HO

OH

OH

HO SucroseOH α-D-glucopyranosyl-(1->2)β-D-fructofuranoside *notice fructose is inverted!!

OH

OH

invertase

OH

fructokinase

OH

OH

Fructose

O

OP

O

Fructose-1-P

aldolase

OH H

O

ADP

OH

H

O

ATP

OP

OH

OP

glyceraldehyde kinase

Glyceraldehyde-3-P

Dihydroxyacetone-P

OH

Glyceraldehyde

Mannose Metabolism OH

ATP

O

OP

ADP

OH HO OH

PO

O

O

OH

OH HO OH

Mannose

hexokinase

OH

OH

Mannose-6-phosphate

Note: Boxed compounds are intermediates of glycolysis.

OH

phosphomannose isomerase or hexose-P-isomerase

OH HO

Fructose-6-P

C. Allen & S. Chen, 7.05 Spring 2001

Glycogen Synthase ATP

ADP

(Glucose)n+1

Overall RXN: Glucose-1-P + (Glucose)n 2 Pi

OH O

O

O

OH

O P O-

OH

O

O-

OH

O

-

O

P

Glucose-1-P

O

O -

P O O

NH

O -

P O

O

UTP OH

OH

O O O

OH

O P O P

OH

O-

OH

O

O -

OH O

NH

O

O

N

O -

N

PPi = O

-

P O

O

O O -

P O

O

-

-

O

H2O O OH

OH

2 Pi = O- P OH

UDPG Uridine-diphospho-glucose

O-

reused in step 1 (Glucose)n Glycogen

UTP

ADP

UDP

ATP

(Glucose)n+1 Glycogen Phosphorylase Pi

OH

(Glucose)n-1

OP O

(Glucose)n

O

OH

Glycogen Phosphorylase

OH OP

OH OH

Glucose-1-P

OH

OH

P-Glucomutase

OH

Glucose-6-P

Note: glycogen phosphorylase cleaves the 1-4 linkage of the non-reducing end of glycogen. The product, glucose-6-P, enters the glycolysis as an intermediate.

C. Allen & S. Chen, 7.05 Spring 2001

Pyruvate Decarboxylase +

operates only under anaerobic conditions (no O2)

C +

readily dissociates (acidic)

S

H

H +

(from Thiamine-PP)

N

(from Thiamine-PP)

N

+

-

CH3

pyruvate

C

S

C

CO2-

CH3

C

S

C α

C

OH

O

.. N

CO2

N

+

N C

β

O

-

CH3

C

S CH3

+

H

S

C

H

H3C

C H

acetaldehyde

:OH ..

OH

O

O C

(looks like a β-keto acid: undergoes spontaneous decarboxylation)

+

H

Pyruvate Carboxylase (Biotin-dependent carboxylation) Bicarbonate = HCO3= hydrated CO2

O

E◊

◊ ◊ C

Biotin

O

O

O

O

◊ ◊C

O

OH

-

P

-

E◊

P

-

O

O O

P

-

O

-

..

HN O

CH2 O

ATP HO

O

P O

(= E-Biotin-CO2)

Pi

O

O

OH

O O

ADP

O -

E◊

Adenosine

from Biotin attached to E

oxaloacetate (OAA)

C

◊ ◊ C

N

HO

NH

-O 2C

CH2

-

CH2

E◊ C

◊ ◊

CO2-

pyruvate

This reaction is technically reversible, but under normal physiological conditions operates in the indicated direction.

CO2-

O

-

C N

O

HO

C O

from Biotin attached to E

C NH

O

O

-

NH

+

H

from Biotin attached to E

C. Allen & S. Chen, 7.05 Spring 2001

PEP Carboxykinase (Biotin-independent decarboxylation) occurs only in liver, not in muscle

oxaloacetate (OAA) O

-

C

β

CH2 α

O

C

C

O

O

O

-

O

O O

-

O

P O

O -

O

P O

O -

H2N

P O

O

CH2

CO2

N

HN

(β-keto acid decarboxylation)

N

N

CH2

*

O

phosphoenol pyruvate (PEP)

-

GDP

GTP HO

OH

* This reaction is technically reversible, but under normal physiological conditions operates in the indicated direction.

O

-

C

C

O

O

P

O

O

-

O

-

C. Allen & S. Chen, 7.05 Spring 2001

Pyruvate Oxidase Particle a.k.a Pyruvate Dehydrogenase Complex

Enzyme

Contains

operates only under aerobic conditions (+ O2)

E1.

Pyruvate Decarboxylase

Thiamine-PP

E2.

Dihydrolipoyl transacetylase

Lipoic Acid, binds CoA

E3.

Dihydrolipoyl dehydrogenase

FAD covalently bound, binds NAD+

Note: The α-Ketoglutarate Oxidase Particle operates via the same mechanism, simply replace pyruvate with α-ketoglutarate E1 ◊

◊ ◊

(from Thiamine-PP) CH3

+

N

C

S

H readily dissociates (acidic)

+

H

(from Thiamine-PP)

E1 ◊

+

◊ ◊

N

-

C

CH3

pyruvate

E1 ◊

CH3

C

CH3

CO2-

O

+

CH3

N

C CH3

E1 ◊

S

Cα

Cβ

+

O

-

S

C

-

C

S

CH3

+

H

S

OH H2C

CH3

N

◊ ◊

C

CH3

CO2

H2C

S

H

E3-FAD+

S

SH CH CH2 E 2

E3-FADH2

CH CH2 E 2 NADH + H+

CH3

C

SCoA

O

acetyl CoA

C OH

-

+

-

S

CoAS

H

S

CH3

E1 -Thiamine-PP

S

H C :OH .. 2

CH CH2 E 2

.. N

◊ ◊

C

C

OH O (looks like a β-keto acid: undergoes spontaneous decarboxylation)

+

◊ ◊

E1 ◊

CH3

N

S

H

E1 ◊

+

◊ ◊

NAD+

CH3

C

S

O H2C

SH CH CH2 E 2

C. Allen & S. Chen 7.05 Recitation Handout Spring 2001

gluconeogenesis

P-enol-pyruvate (PEP) C

NADH + H+

CO2-

only in yeast and some microorganisms

pyruvate

ATP

ADP

CH3

anaerobic conditions CO2(no O2)

C

pyruvate kinase

O

OP

C

GDP PEP carboxykinase

pyruvate carboxylase

*

*

GTP

-O2C

CH2

C

CH2

NAD+

lactate CH3

lactate dehydrogenase

CH

CO2-

OH S-CoA

NAD

oxaloacetate (OAA)

alcohol dehydrogenase

OH

NADH + H+

+

ADP

CH3

pyruvate decarboxylase

ATP

HCO3-

ethanol

H

O

aerobic conditions (+ O2)

CO2

NAD+

acetaldehyde CH3 CO2

glycolysis pathway

CH2

The Diverse Fates of Pyruvate

Thiamine-PP, Lipoate, FAD

**

CO2

pyruvate oxidase particle (a.k.a. pyruvate dehydrogenase complex)

glyoxylate cycle can occur only in microorganisms NOT in animals!

NADH

acetyl CoA CH3

CO2-

C

S-CoA

O

O

H2O citrate synthase

Krebs TCA Cycle

S-CoA

***

* ** ***

This reaction is technically reversible, but under normal physiological conditions operates in the indicated direction. Cycle can form a net amount of OAA from acetyl CoA OAA is regenerated, but no net OAA can be formed from acetyl CoA

citrate HO

CH2

CO2-

C

CO2-

CH2

CO2-

Victor Sai 7.05 Spring 2004

Playing the Pathway Game 1) Identify the starting materials and the products. Convert molar ratios to whole numbers (e.g., 0.004 M would be equivalent to 4 mols) 2) Determine which cycles are available. (a) Glyoxylate Cycle only available to plants and microorganisms. Liver tissue means Glyoxylate Cycle is not available. (b) Anaerobic conditions (no O2): cannot enter TCA cycle, pyruvate converted to lactate (in all organisms) or ethanol (in yeast and some microorganisms). 3) Make note of any inhibitors. (a) ARSENITE Blocks pyruvate oxidase particle & α-ketoglutarate oxidase particle (b) ARSENATE Analog of phosphate; incorporated by glyceraldehyde-3-P dehydrogenase in glycolysis, leading to NO ATP PRODUCTION from glycolysis (c) MALONATE Blocks converstion between succinate and fumarate in TCA cycle by inhibiting succinic dehydrogenase

COUNTING ATP 1 cytosolic NADH (only in glycolysis) = 2 ATP 1 mitochondrial NADH = 3 ATP 1 FADH2 = 2 ATP 1 GTP = 1 ATP NET GAIN OF 1 OAA PER RUN THROUGH THE GLYOXYLATE CYCLE (each run starts with 2 Acetyl CoA)

Page 1 of 2

Note: Mechanism for Glyceraldehyde-3-P Dehydrogenase Mechanism for Forward Reaction given in class. Mechanism for Reverse Reaction: (1,3-di P Glycerate O E NAD

S+

Glyceraldehyde-3-P Dehydrogenase

H

Glyceraldehyde-3-P) O

O

C

OP

E

C

OH

NAD

CH2OP 1,3-di P Glycerate

S +

C H

C

NAD+

NADH

E

S

NADH H

OH

C C

OH

CH2OP

CH2OP

E

CHO H

C

OH

NAD

S+

CH2OP Glyceraldehyde-3-P

Page 2 of 2

Carbohydrate Metabolism

Victor Sai 7.05 Spring 2004

Glucose Glucose Lactose

β-galactosidase

ATP

Galactose ATP ADP

Galactokinase

Sucrose

Fructose

Invertase

ADP

Fructokinase

Dihydroxyacetone-P

Fructose-1-P

Aldolase

Glyceraldehyde

UDP-Glucose

Pi (Glucose)n (Glycogen)

(Glucose)n-1

epimerase

Galactose-1-P Uridylyl Transferase

UDP-Galactose UTP PPi

Glucose-1-P

Glycogen Phosphorylase

Glucose

UDP-Glucose

Glucose-6-P

Glucose-6-P phosphatase

ATP Mannose

ADP

Hexokinase

Mannose-6-P

s xo He

e-P

Iso

me

ras

e

Pi

UTP

Fructose-1,6-di P

Aldolase

(2) 3-P-Glycerate

HCO3-

Pyruvate Carboxylase

PEP Carboxykinase

Malate

NADH + H+

NADH

(2) Oxaloacetate

c c

c

Malate Dehydrogenase

+

(2) NADH + H (2) NAD+

m Fu

Fumarate

Fumarate ADP

FADH2

ATP

GTP

TCA Cycle

e S De u c c hy i ni dr c og en

FADH2

Succinic Dehydrogenase

FAD

as ar

CoA H2 O

CoA

Succinate Succinic Thiokinase

H2 O

H2 O

Malate Synthase

Acetyl AMP

PPi

ATP

Acetate Thiokinase

(2) Ethanol Alcohol Dehydrogenase

Acetate

H2 O cis-Aconitate

Citrate

Citrate Synthase

Aconitase

H2 O Aconitase

H2 O

Isocitratase

Aconitase

cis-Aconitate H2 O

Glyoxylate Cycle

Isocitrate

Aconitase

Isocitrate

NAD+

Succinate

Isocitrate Dehydrogenase

CO2 Succinyl CoA

+ (2) NADH+H+ (2) NAD

(2) Acetaldehyde

CoA

CoA Citrate

(2) 2-P-Glycerate Enolase

SCoA

Acetate Thiokinase

e

FAD GDP+Pi

c c c

Glyoxylate as

(2) CO2

CO2 AMP (2) Acetyl CoA

(2) Malate

Pyruv ate Decarb oxylas e

SCoA Pyruvate Oxidase Particle

Malate Dehydrogenase

H 2O

Fumarase

H2 O

NAD+

(2) P-Enol-Pyruvate

Pyruvate Kinase

P-Glyceromutase

(2) H2O

(2) ATP (2) ADP

(2) Pyruvate

(2) ADP

(2) ADP (2) ATP

P-Glycerokinase

CO2

(2) ATP

(2) 1,3-di P-Glycerate

Dihydroxyacetone-P

Glycolysis

NAD+

(2) Pi (2) NAD+ (2) NADH Glyceraldehyde-3P

H2 O

GTP

ADP

Dehydrogenase Triose-P Isomerase

Fructose-1,6-diP phosphatase

GDP

ATP

Glyceraldehyde-3-P

ADP

Fructose-6-P P-Fructokinase

Hexose-P Isomerase

UDP

Glycogen Synthase

ATP

ADP

Hexokinase

ATP ADP

(Glucose)n (Glucose)n+1

P-Glucomutase

ATP

Glyceraldehyde Kinase

Galactose -1-P

CoA α-ketoglutarate

NADH Oxidase Particle

CO2

α-ketoglutarate NAD+

NADH + H+

Oxalosuccinate