Glucose + Protein Respiration and ATP

Question 1

Define catabolic and anabolic reactions

Answer 1

The energy contained in phosphoanhydride bond is a way for the cell to harness the energy liberated from breaking bonds within the food molecules in catabolic reactions. The backbones of the molecules are also useful starting points to synthesize components needed for the cell, known as anabolic reactions.

Question 2

What are the 3 stages of cellular metabolism?

Answer 2

1. Glycolysis: oxidation of glucose in cytosol to form ATP and NADH
2. TCA or Krebs Cycle: further oxidation of small molecules in the mitochondria to produce NADH, ATP and FADH2
3. Oxidative Phosphorylation: generation of ATP in the mitochondria through the reduction of O2 and H2O (and the reoxidation of the products aformentioned)

Question 3

What are the 6 reactions that make up cell metabolism?

Answer 3

1. Redox: Electron transfer reactions
2. Ligation requiring ATP cleavage: Formation of covalent bonds
3. Isomerization: Rearrangement of atoms to form isomers
4. Group transfer: Transfer of functional group from one molecule to another
5. Hydrolytic: Cleavage of bonds by addition of water
6. Addition/removal of functional groups: Addition of functional groups to double bonds or their removal to form double bonds.

Question 4

What are the 2 stages of glycolysis?

Answer 4

1. Formation of high energy compound using investment of ATP
2. Splitting of high energy compounds to produce useful energy in form of ATP

Question 5

Describe first stage of glycolysis: TRAP, SHAPE, KINASE, ALDOLASE, TPI

Answer 5

1. Formation of glucose-6-phosphate from glucose catalysed by hexokinase. Irreversible reaction so commits molecule to process and traps glucose in cell as phosphate has negative charge.
2. Glucose-6-phosphate to fructose-6-phosphate catalysed by phosphoglucose isomerase. Fructose can be split into equal halves when cleaved.
3. Fructose-6-phosphate to fructose-1,6-biphosphate catalysed by phosphofructokinase. Key regulation step for entry into glycolytic pathway so enzyme heavily regulated.
4. Fructose-1,6-biphosphate hydrolysed by adolase to form glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
5. Dihydroxyacetone phosphate converted to glyceraldehyde-3-phosphate by triose phosphate isomerase. Only glycolytic enzymopathy that can be fatal with prognosis of 6 years.

Question 6

Describe second stage of glycolysis-SURPRISE TOOL, REVERSE PHOSPHOR., MUTASE, ENOLASE, KINASE

Answer 6

6. Glyceraldehye-3-phosphate converted to 1,3-bisphosphoglycerate by glyceraldehye-3-phosphate dehydrogenase. NADPH formed while ATP broken down.
7. 1,3-bisphosphoglycerate converted to 3-phosphoglycerate by phosphoglycerate kinase. ATP formed.
8. 3-phosphoglycerate converted to 2-phosphoglycerate by a phosphoglycerate mutase.
9. Enolase acts by removing a water group from 2-phosphoglycerate forming phosphoenolpyruvate
10. Phosphoenolpyruvate acted on by pyruvate kinase to remove a phosphate group forming ATP and pyruvate. Irreversible reaction.

Question 7

What is the net result of glycolysis?

Answer 7

2 ATP, 2 NADH, 2 pyruvate

Question 8

What are the 3 fates of pyruvate?

Answer 8

Alcohol fermentation of two forms and generation of lactate. Pyruvate forms acetaldehyde when acted on by pyruvate decarboxylase and acetaldehyde forms ethanol when acted on by alcohol dehydrogenase. Pyruvate forms lactate when acted on by lactate dehydrogenase. Pyruvate forms acetyl CoA and carbon dioxide when acted on by the pyruvate dehydrogenase complex.

Question 9

When does alcohol fermentation happen?

Answer 9

Occurs mainly in yeast under anaerobic conditions

Question 10

When does generation of lactate happen and why?

Answer 10

Occurs mainly in mammalian muscle during intense exercise when oxygen is a limiting factor. Is anaerobic. Regenerates NAD allowing glycloysis to continue.

Question 11

How is acetyl CoA generated and used?

Answer 11

Generated as pyruvate is oxidised to acetyl CoA by pyruvate dehydrogenase complex and hence committed to TCA cycle. Contains a thioester bond which is a high energy bond and so easy to break. Thus, acetate group can be donated to any other molecule.

Question 12

What is Beri-Beri disease and how is it caused?

Answer 12

Caused by a deficiency in thiamine (derivative of vitamin B1 and co-factor of PDH complex). Symptoms include damage to PNS, weakness of musculature and decreased cardiac output. Brain heavily relies on glucose metabolism so especially vulnerable.

Question 13

What is the net result of the TCA cycle?

Answer 13

3 NADH, 1 GTP, 1 FADH2 and 2 CO2

Question 14

Why does the Krebs cycle operate under aerobic conditions?

Answer 14

Krebs cycle enzymes with one exception are located in mitochondrial matrix and majority of ATP generated when co-enzymes reduced in this step are reoxidised with help of O2 in oxidative phosphorylation.

Question 15

How do amino acids interact with TCA cycle?

Answer 15

Most are fed into TCA cycle at various stages or involved in forming glucose. Ketogenic amino acids are fed into making acetyl-coenzyme A or acetoacetyl-coenzyme A while glucogenic amino acids are fed in at other stages. There are only 7 products that all amino acids can feed into: Pyruvate, Acetyl CoA, Acetoacetyl CoA, alpha-ketoglutarate, Succinyl CoA, Fumarate and Oxaloacetate.

Question 16

What are transamination reactions? Give an example

Answer 16

Transamination reactions are group transfer reactions in which an amine group is transferred from an amino acid to a keto acid to form a new amino acid and keto acid. An example is alanine metabolism. Alanine transfers its amino group to alpha keto glutarate, forming pyruvate and glutamate. The pyruvate goes into the TCA cycle and the glutamate is made back into alpha keto glutarate, forming NH4+ which is eventually converted to urea.

Question 17

Why are shuttles used? What shuttles are used and where?

Answer 17

Glycolysis occurs in the cytosol, however the NADH produced is needed in the mitochondria to be used in oxidative phosphorylation and to regenerate NAD+.
There is a finite amount of NAD+ so failure to regenerate would lead to glycolysis stopping thus it is transported via shuttles. Skeletal muscle and brain mainly use the glycerol phosphate shuttle whereas the liver, kidney and heart use the malate-aspartate shuttle.

Question 18

How does the glycerol phosphate shuttle work?

Answer 18

There are two versions of the same enzyme, cytosolic and membrane bound mitochondrial. The cytosolic glycerol 3-phosphate dehydrogenase transfers electrons from NADH, forming NAD+, to dihydroxyacetone phosphate forming glyceraldehyde 3-phosphate. This then binds to the membrane-bound enzyme glycerol 3-phosphate which takes the electrons from the glyceraldehyde and passes them onto FAD to form FADH2. The FADH2 then passes this onto co-enzyme Q and hence the electrons enter the electron transport chain.

Question 19

How does the malate-aspartate shuttle work?

Answer 19

Two enzymes are involved in this reaction as redox and transamination reactions occur. Cytosolic aspartate undergoes a transamination reaction catalysed by aspartate transaminase to form oxaloacetate as alpha KG is formed into glutamate. This oxaloacetate undergoes a redox reaction to form malate catalysed by malate dehydrogenase, where electrons from NADH are transferred to malate. The opposite occurs inside the mitochondrial matrix. The two anti-porters allow glutamate-aspartate on one hand and malate-alpaKG on the other hand to move across the membrane.

Question 20

What is the net yield of the Krebs Cycle

Answer 20

1 Acetyl CoA gives: 3 NADH, 1 FADH2, 1 GTP and thus 12 ATP. 1 NADH gives 3 ATP and 1 FADH2 gives 2 ATP.

Question 21

What are some TCA cycle defects that can cause cancer?

Answer 21

Mutations in the genes Isocitrate Dehydrogenase, Succinate Dehydrogenase and Fumerase can cause cancer as they decrease TCA activity and hence enhance aerobic glycolysis. This is when glucose forms lactate even when enough O2 is present known as the Warburg Effect.

Question 22

Which amino acids can be phosphorylated?

Answer 22

Serine, threonine and tyrosine as they all contain am OH group

Question 23

What are the types of phosphorylation?

Answer 23

Substrate level phosphorylation is where there is a direct transfer of a phosphate group to ADP to form ATP. Oxidative phosphorylation is where there is generation of ATP from an electron transport system.

Question 24

Describe structure of mitochondria

Answer 24

They have an outer membrane (which limits the size of the organelle) and an inner membrane (folds that project inward called cristae). The reactions of oxidative phosphorylation take place in the inner membrane, in contrast to the Krebs Cycle reactions which occur in the matrix. Numerous folds within the cristae increase the surface area upon which oxidative phosphorylation can take place.

Question 25

Why is redox involving co-factors as effective way to harness energy?

Answer 25

The energy released from the re-oxidation of NADH and FADH2 is enough to generate several phosphoanhydride bonds and much of this is recovered from the components of the electron transport chain to synthesise ATP.

Question 26

What is the significance of mtDNA?

Answer 26

Human mitochondrial DNA codes for only 37 genes but is very important as mutations are known to frequently be the cause of disease. It's passed on via the ovum thus is inherited from the mother. mtDNA is present within cells in multiple copies, some of which are mutated and some which are not (heteroplasmy)

Question 27

Why are mitochondria considered evolutionary descendents of prokaryotes in an endosymbiotic relationship with eukaryotic cell ancestors?

Answer 27

1. Mitochondria only arise from pre-existing mitochondria or chloroplasts in plants' 2. Have their own genome, resembling prokaryotes with single DNA loop and no histones 3. Number of antibiotics that act by blocking bacterial protein synthesis also block protein synthesis in mitochondria 4. Have own protein synthesisng machinery 5. First amino acid of mitochondrial transcript is fMet (formylated Methionine) unlike eukaryotic Met.

Question 28

What does the electron trasnport chain consist of? What does it do?

Answer 28

``` Four enzymes: NADH Dehydrogenase complex (Complex I) Succinate Dehydrogenase complex (Complex II) Cytochrome b-c1 complex (Complex III) Cytochrome oxidase complex (Complex IV) Two carriers: Ubiquinone (co-enzyme Q) Cytochrome C ``` These proteins accept electrons and thus a proton (H+) from the aq solution. As electrons pass through each of the complexes, a proton is passed or ‘pumped’ to the intermembrane space

Question 29

Why does FADH2 produce less ATP per molecule?

Answer 29

Succinate dehydrogenase (Complex II) is an integral membrane protein that is firmly attached to the inner surface of the inner mitochondrial membrane. There, it can communicate directly with ubiquinone. As such one less proton is pumped to the intermembrane space, c.f. NADH and as a consequence, less ATP is produced

Question 30

Define redox reactions

Answer 30

Redox (Reduction-Oxidation) reactions are defined as electron transfer reactions involving a reduced substrate (which donates electrons and therefore becomes oxidised) and an oxidised substrate (or oxidant) which accepts electrons and becomes reduced in the process.

Question 31

Define reduction potential

Answer 31

The ability of a redox couple to accept or donate electrons can be determined experimentally and is known as the reduction potential or redox potential. Standard redox potentials (E’0) can be measured experimentally. A negative E’0 implies that the redox couple has a tendency to donate electrons and therefore has more reducing power than hydrogen. Conversely, a positive E’0 implies that the redox couple has a tendency to accept electrons and therefore has more oxidising power than hydrogen.

Question 32

Describe ATP synthase and how it acts

Answer 32

ATP synthase is a multimeric enzyme consisting of a membrane bound part (F0) and a F1 part which projects into the matrix space. F0 is called F0 or FO because it is inhibited by oligomycin. Rotation of the enzyme drives transitions states, with altering affinities for ATP and ADP. As a consequence, conformational energy flows from the catalytic subunit into the bound ADP and Pi to promote the formation of ATP (chemical energy). Depending on the direction of the flow of protons through the ATP synthase, the complex can either generate ATP or consume it

Question 33

How does the oxygen electrode work?

Answer 33

The Oxygen Electrode is a device that measures the [oxygen] in a solution housed within a small chamber. The base of the chamber is formed by a teflon membrane permeable to oxygen. Under the membrane is a compartment containing two electrodes, a platinum (Pt) cathode and a silver (Ag) anode. A small voltage of around 0.6 volts is applied between the anode (+) and cathode (-). Oxygen diffuses through the teflon membrane and is reduced to water at the platinum cathode. The circuit is completed at the silver anode, which is slowly oxidised to AgCl by the KCl electrolyte. The resulting current is therefore proportional to the oxygen concentration in the sample chamber.

Question 34

Why is the oxygen electrode used?

Answer 34

We can use the oxygen electrode to dissect various components of the electron transport chain. The first step is to prepare a suspension of mitochondria from a tissue and place them into the chamber of the oxygen electrode. If the oxygen consumption of the suspension is then monitored for a set time period, the effects of various substrates and inhibitors on the electron transport chain can be determined.

Question 35

What is respiratory control?

Answer 35

Uptake of oxygen by mitochondria is controlled by the components of ATP production: Inorganic phosphate (Pi), and ADP. This is known as respiratory control and allows the body to adapt oxygen consumption to actual energy requirements.

Question 36

What are metabolic poisons?

Answer 36

Molecules that interfere with either: The flow of electrons along the ETC or the flow of protons through ATP synthase. By definition, interrupt ATP synthesis. As such, they are highly toxic and are termed metabolic poisons

Question 37

How do cyanide (CN-) and azide (N3-) function?

Answer 37

Bind with high affinity to the ferric (Fe3+) form of the haem group in the cytochrome oxidase complex (IV) blocking the final step of the ETC.

Question 38

How does malonate function?

Answer 38

Closely resembles succinate and acts as a competitive inhibitor of succinate dehydrogenase. It slows down the flow of electrons from succinate to ubiquinone by inhibiting the oxidation of succinate to fumarate.

Question 39

How does rotenone function?

Answer 39

An isoflavone found in the roots and seeds of some plants. It inhibits the transfer of electrons from complex I to ubiquinone.

Question 40

How does oligomycin function?

Answer 40

An antibiotic produced by Streptomyces that inhibits oxidative phosphorylation by binding to the ‘stalk’ of ATP synthase and blocking the flow of protons through the ATP Synthase.

Question 41

How does dinitrophenol function?

Answer 41

Is a proton ionophore which can shuttle protons across the inner mitochondrial membranes.

Question 42

How did dinitrophenol function as a weight loss drug?

Answer 42

Can induce weight loss by transporting protons across the mitochondrial membrane. It was shown to cause weight loss by uncoupling oxidative phosphorylation, leading to a heightened metabolic rate and increased fat metabolism. The rapid consumption of calories was thought to occur because of the shift in the proton electrochemical gradient, which results in potential energy from the proton motive force dissipating as heat, instead of being converted to ATP. This mechanism of action also leads to an accumulation of pyruvate and lactic acids. Unlike thyroid hormone, which was used for weight loss, it did not impact on nitrogen excretion and thus was postulated to cause fat rather than lean muscle mass loss.

Question 43

What were the side effects of dinitrophenol?

Answer 43

The resultant excess heat production led to uncontrolled hyperthermia following failed mechanisms of thermoregulatory homeostasis, which results in significant morbidity and mortality.11 Within a few years of its use, several adverse effects including toxic hyperthermia, hepatotoxicity, formation of cataracts, and few cases of agranulocytosis were reported.

Question 44

What is non-shivering thermogenesis?

Answer 44

In response to a drop in core body temperature, thermogenin (or UCP-1) is activated, which similarly uncouples respiration from ATP and releases heat from the dissipation of the proton gradient.