Lecture 19

Question 1

where does cellular respiration occur?

Answer 1

• Glycolysis – cytosol

* Respiration – mitochondria

Question 2

where does ATP generation occur?

Answer 2

inner membrane of mitochondrion

Question 3

where does pyruvate oxidation and CAC occur?

Answer 3

matrix of mitochondrion

Question 4

cristae

Answer 4

folds in the inner mitochondrial membrane that project into the mitochondrial matrix. The enzymes of the ETC and oxidative phosphorylation are located mainly on the cristae

Question 5

how is pyruvate transported from the cytosol into the mitochondria? and what is it converted to?

Answer 5

• mitochondrial pyruvate carrier (MPC)
• converted to acetyl CoA
• pyruvate is a “mono carboxylate”

Question 6

rxn for conversion to acetyl CoA

Answer 6

pyruvate + CoA + Nad+ —> Acetyl CoA + CO2 + NADH

Question 7

what is an oxidative decarboxylation catalyzed by?

Answer 7

pyruvate dehydrogenase complex

Question 8

what family does pyruvate dehydrogenase complex belong to?

Answer 8

family of α-keto Dehydrogenases

Question 9

what is the ΔGº’ of the pyr–> CoA rxn?

Answer 9

Pyruvate + CoA + NAD+ —> Acetyl CoA + CO2 + NADH

-33.5 kJ/mol

Question 10

What three enzymes catalyze the 5 rxns

Answer 10

E1- pyruvate dehydrogenase
E2- dihydrolipoamide transacetylase
E3- Dihydrolipoamide dehyrdogenase

Question 11

The 3 enzymes require which 5 cofacors?

Answer 11

• Thiamine Pyrophosphate (TPP)
• Lipoic Acid
• Coenzyme A (CoA or CoA-SH)
• Flavin Adenine Dinucleotide (FAD)
• Nicotinamide Adenine Dinucleotide (NAD+)

Question 12

which cofactor stabilizes carboanions?

Answer 12

thiamine pyrophosphate

thiamine (vitamin b1)

Question 13

The 3 enzymes require which 5 cofactors?

Answer 13

• Thiamine Pyrophosphate (TPP)
• Lipoic Acid
• Coenzyme A (CoA or CoA-SH)
• Flavin Adenine Dinucleotide (FAD)
• Nicotinamide Adenine Dinucleotide (NAD+)

Question 14

which cofactor stabilizes carboanions?

Answer 14

thiamine pyrophosphate

thiamine (vitamin b1

Question 15

what are coenzymes?

Answer 15

Coenzymes are small molecules. They cannot by themselves catalyze a reaction but they can help enzymes to do so. In technical terms, coenzymes are organic nonprotein molecules that bind with the protein molecule (apoenzyme) to form the active enzyme (holoenzyme)

Question 16

Pyruvate Dehydrogenase (PDH) 
what are the methods to identify

Answer 16

is a large multisubunit complex.

Use Crystallography and NMR to solve structures of domains, subunits, and enzymes

Use Electron Microscopy to determine how they are organized

Not easy – Complex is somewhat flexible

Question 17

Pyruvate Dehydrogenase Complex

What’s the advantage?

Answer 17

• Substrate Channeling- int. of multistep pathway are handed off form one active site to the next w/o diffusing complex- makes smooth
• Effective Concentration-
• A + B <==> A-B + A-B
• tethering can increase the effective concentration

• Regulation-

Question 18

TPP- Location and function

Answer 18

• tightly bound to E1

- decarboxylates pyruvate, yielding hydroxyethyl- TPP

Question 19

Lipoic Acid- location and function

Answer 19

• covalently bound to E2 via lysine (“swinging arm”)

- accepts hydroxyl carbanion from TPP as acetyl group

Question 20

Coenzyme A (CoA)- location and function

Answer 20

• dissociable substrate for E2

- accepts acetyl group from lipoamide

Question 21

FAD- location and function

Answer 21

• tightly bound to E3

- accepts pair of e- from reduced lipoamide

Question 22

NAD+ - location and function

Answer 22

• dissociable substrate for E3

- accepts pair of e- from reduced FADH2

Question 23

Enzymes of the Krebs cycle

Answer 23

Pyruvate Dehydrogenase & α-Ketoglutarate Dehydrogenase are in the same enzyme family.

Question 24

ReWhat is PDH inhibited by?

Answer 24

acetyl CoA, NADH

compete for binding with substrates CoA and NAD+

Question 25

what is PDHG regulated by?

Answer 25

covalent modifcation: • PDH kinase (inhibits) (phosphorylates E1) –kinase activated by NADH and acetyl CoA –kinase inhibited by NAD+ and pyruvate • PDH phosphatase (activates) –phosphatase activated by insulin and Ca2+

Question 26

Transcriptional regulation of PDH kinase

Answer 26

Expression of many glucose metabolism genes is regulated by a specific transcription factor HIF-1. low O2--> stabilized HIF --> DNA --> RNA--> Protein --> PDH Kinase

Question 27

what is HIF-1 and what is it stabilized by?

Answer 27

transcriptional factor | hypoxia

Question 28

hydroxylation

Answer 28

is a signal to degrade HIF-1 by a proteolysis pathway (ubiquitin pathway)

Question 29

HYPOXIA (low O2) --> HIF1 stabilized low or high ``` PDH PDH activity pyruvate consumed acetyl CoA made glycolytic enzymes CAC activity ```

Answer 29

``` More PDH Kinase PDH Phosphorylation Increases PDH Activity Decreases Less Pyruvate is consumed Less Acetyl CoA is made More glycolytic enzymes made Less Citric Acid Cycle Activity ```

Question 30

More lactic acid production rxn:

Answer 30

Pyruvate + NADH ----> Lactate + NAD+

Question 31

Hypoxia-Inducible Factor (HIF) is important for:

Answer 31

-(HIF-1 also stimulates production of specific types of Growth Factors (VEGF and EPO)) •Adaptation to high altitudes •Survival and growth of cells in low O2 environments. •Tumor growth

Question 32

VEGF

Answer 32

(Vascular Endothelial Growth Factor and Erythropoietin) VEGF stimulates formation of new blood vessels. • EPO stimulates production of new erythrocytes

Question 33

Erythropoietin (“EPO”)

Answer 33

* made in kidneys (and liver) * heavily glycosylated * stimulates survival of red blood cell progenitors The “JAK/STAT” pathway is another type of signaling pathway that uses reversible phosphorylation of proteins.

Question 34

high altitude training

Answer 34

“Through the production of normobaric hypoxic (oxygen reduced) air, we can simulate altitudes of up to 21,000ft/6,400m. As a result, athletes, fitness enthusiasts and health conscious individuals worldwide can take advantage of the benefits associated with altitude training while at sea-level”.

Question 35

too much EPO can be dangerous - WHY?

Answer 35

increase the thickness (viscosity) of the blood, raising the risk for blood clots, heart attacks, and strokes

Question 36

key concepts for PDH- Regulation

Answer 36

• Regulation – Inhibition by products (NADH and Acetyl CoA) – Phosphorylation of Pyruvate Dehydrogenase – Regulation of PDH Kinase – Regulation of expression – Mechanism by which HIF senses Oxygen

Question 37

When glucose is converted to ethanol by yeast (as in the production of beer) how much of the chemical energy that was originally stored in glucose still remains in the end product, ethanol, that is produced?

Answer 37

92%

Question 38

Which one of the following molecules has the highest molecular weight?

Answer 38

Acetyl CoA

Question 39

When Acetyl CoA reacts with oxaloacetate in the Citrate Synthase reaction how many carbons are in the product, citrate that is formed?

Answer 39

5

Question 40

Which ONE of the following is NOT a coenzyme or substrate involved in the pyruvate dehydrogenase complex reaction?

Answer 40

ATP`

Question 41

Which ONE of the following provides a long arm that can swing a substrate from one site to another in the PDH complex?

Answer 41

Lipoamide

Question 42

To get you to look carefully at these structures – answer this - which ONE has the highest molecular weight?

Answer 42

FAD

Question 43

Which part of the PDH complex is affected most directly by Arsenite?

Answer 43

FAD