Carbohydrates, CAC & Terminal Respiration Flashcards

Question

What are the benefits of having carbohydrates attached to proteins?

Answer 1

Carbohydrates attached to proteins may, – Increases the proteins solubility – Influence protein folding and conformation – Protect it from degradation – Act as communication between cell

Answer 2

1. GAGs = Glycosaminoglycans 2. They used to be called mucopolysaccharides. 3. Found in mucus and also synovial fluid around joints. 4. Un-branched polymers made from repeating units of hexuronic acid and an amino-sugar, which alternate through the chains 5. NOTION 1.9

Answer 3

1. Carbohydrates >> protein 2. Formed from GAGs covalently attaching to proteins. 3. They are macromolecules found on the surface of cells or in between cells in the extracellular matrix. Therefore form part of many connective tissues in the body.

Answer 4

Usually found on the outer plasma membrane and extra cellular matrix, but also in the blood and within cells in the secretory system (Golgi complex, secretory granules). Some cytoplasmic and nuclear proteins are also glycoproteins.

Answer 5

1. Group of genetic disorders caused by the absence or malfunction of enzymes that are required for the breakdown of glycosaminoglycans. 2. Over time the glycosoaminoglycans build up in connective tissue, blood and other cells of the body 3. Can cause severe dementia, problems with the heart and any other endothelial structure as the glycosaminoglycans build up between the endothelial cells. Also, bones tend to be stunted and joints will be inflammed and become severely damaged. 4. Hurler, Scheie, Hunter, Sanfilippo syndromes are a few mucopolysaccharidoses

Answer 6

Severe developmental defects: – Stop developing at around 4 years – Death at around 10 years old Clouding and degradation of the cornea Arterial wall thickening. Dementia caused by, amongst other things: – Build up of CSF – Enlarged ventricular spaces Experimental therapies currently include: – Gene therapy – Enzyme replacement therapies

Answer 7

Starch: Cereals, potatoes, rice Glycogen: Meat (however when the animal dies enzyme activity in the tissue degrades much of the glycogen stores) Cellulose and hemicellulose: Plant cell walls – we don’t digest this Oligosaccharides containing (α1→6) linked galactose: Peas, beans, lentils – not digested Lactose, sucrose, maltose: Milk, table sugar, beer Glucose, fructose: Fruit, honey

Answer 8

Mouth: Salivary amylase hydrolyses (alpha 1-4) bonds of starch.

Answer 9

Stomach: No carbohydrate digestion

Answer 10

Duodenum: Pancreatic amylase works as in mouth

Answer 11

1. Jejunum = Final digestion by mucosal cell-surface enzymes 2. a. Isomaltase - Hydrolyses (alpha 1-6) bonds b. Glucoamylase (removes Glc sequentially from non reducing ends) c. Sucrase - Hydrolyses sucrose d. Lactase - Hydrolysis lactose

Answer 12

- Glucose is absorbed through an indirect ATP-powered process - ATP-driven Na+ pump maintains low cellular [Na+], so glucose can continually be moved in to the epithelial cells - This system continues to work even if glucose has to be moved into the epithelial cells against it’s concentration gradient (i.e. When blood glucose is high) NOTION 1.10

Answer 13

Galactose has a similar mode of absorption as glucose, utilising gradients to facilitate it’s transport.

Answer 14

Fructose is slightly different from glucose/ galactose: – Binds to the channel protein (GLUT5) – Simply moves down it’s concentration gradient (high in gut lumen, low in blood)

Answer 15

These cannot be digested by the gut, but they do have a use – Increase faecal bulk and decrease transit time. Polymers are broken down by gut bacteria – Yielding CH4 and H2 Beans will also have the same effect!

Answer 16

Deficiencies may be genetic Or, they can result from, – Severe intestinal infection – Other inflammation of the gut lining – Drugs injuring the gut wall – Surgical removal of the intestine Characterised by abdominal distention and cramps. Diagnosis would require enzyme tests of intestinal secretions – Usually checking for lactase, maltase or sucrase activity

Answer 17

Most humans lose lactase activity after weaning Western whites retain lactase activity into adulthood. Theory that this comes from cattle domestication 100,000 years ago.

Answer 18

If lactase is lacking, then ingestion of milk will give disaccharidase deficiency symptoms. This happens for 2 reasons: – Undigested lactose is broken down by gut bacteria causing gas build up and irritant acids – Lactose is osmotically active, thus drawing water from the gut into the lumen causing diarrhoea

Answer 19

Symptoms can be avoided by, – Avoiding milk products (many non-western diets do) – Using milk products treated with fungal lactase – Supplementing diet with lactase

Answer 20

1. Glc diffuses through the intestinal epithelium cells into the portal blood and on to the liver. 2. Glc is immediately phosphorylated into glucose 6-phosphate by the hepatocytes (or any other cell glucose enters). 3. Glucose 6-phosphate cannot diffuse out of the cell because GLUT transporters won’t recognise it – This effectively traps the glucose in the cell

Answer 21

Enzyme catalyst, – Glucokinase (liver) – Hexokinase (other tissues)

Answer 22

- Blood [Glc] normal – the liver doesn’t “grab” all of the glucose, so other tissues have it - Blood [Glc] high (after meal) - liver “grabs” the Glc - High glucokinase Vmax means it can phosporylate all that Glc quickly, thus most absorbed Glc is trapped in the liver - Hexokinase low Km means even at low [Glc] tissues can “grab” Glc effectively - Hexokinase low Vmax means tissues are “easily satisfied”, so don’t keep “grabbing” Glc

Answer 23

NOTION 2.1

Answer 24

~90% is in the liver and skeletal muscle

Answer 25

In the liver: – [blood Glc] falls, glycogen -> G-6-P -> Glc into the blood In skeletal muscle: – there is no glucose 6-phosphatase, glycogen -> G-6-P -> lactate

Answer 26

1. Glycogenin begins the process by covalently binding Glc from uracildiphosphate (UDP)- glucose to form chains of approx. 8 Glc residues 2. Then glycogen synthase takes over and extends the Glc chains 3. The chains formed by glycogen synthase are then broken by glycogen-branching enzyme and re-attached via (α1→6) bonds to give branch points

Answer 27

1. Glc monomers are removed one at a time from the non-reducing ends as G-1-P (by glycogen phosphorylase) 2. Following removal of terminal Glc residues to release G-1-P, by glycogen phosphorylase, Glc near the branch is removed in a 2-step process by debranching enzyme 3. Transferase activity of de-branching enzyme removes a set of 3 Glc residues and attaches them to the nearest non-reducing end via a (α1→4) bond 4. Glucosidase activity then removes the final Glc by breaking a (α1→6) linkage to release free Glc 5. This leaves an unbranched chain, which can be further degraded or built upon as needed NOTION 2.2

Answer 28

Von Gierke’s Disease = Liver (and kidney, intestine) glucose 6-phosphatase deficiency

Answer 29

Symptoms: – High [liver glycogen] – maintains it’s normal structure – Low [blood Glc] – fasting hypoglycaemia - This is because glycogen cannot be used as an energy source – all Glc must come from dietary carbohydrate – High [blood lactate] – lacticacidaemia - Because the lactate produced by skeletal muscle cannot be reconverted to Glc in the liver

Answer 30

Regular carbohydrate feeding – little and often - Every 3-4 hours throughout the day and night - Can be administered through a nasogastric tube and pump, but sudden death has occurred when the pump fails or the tube disconnects

Answer 31

Skeletal muscle phosphorylase deficiency

Answer 32

Symptoms: – High [muscle glycogen] – maintains it’s correct structure – Weakness and cramps after exercise – No increase in [blood glucose] after exercise Most symptoms are not apparent in resting state, when muscles will use other energy sources (Glc and fatty acids from the blood).

Answer 33

Usually becomes apparent in 20-30 year olds – Children do suffer the disease but may remember pain during adolescence and childhood

Answer 34

1. Avoid strenuous activity 2. Make use of your “second wind” - Exercise briefly (anaerobically), wait for the pain to subside, continue to exercise (aerobically using oxidative phosphorylation of fatty acids)

Answer 35

It is essentially the only way that energy can be made from fuel molecules when cells lack O2 (exercising muscle) or mitochondria (RBCs).

Answer 36

There are 2 main phases of glycolysis: - Preparatory Phase - Payoff Phase For each Glc, 2 ATP are used in the preparatory phase and 4 ATP gained in the payoff phase.

Answer 37

For 1 Glc passing through the preparatory phase: 2 molecules of G-3-P formed to enter the payoff phase.

Answer 38

Thus glycolysis gives a net gain of 2 ATP (and NADH) per Glc molecule.

Answer 39

10 stages of Glycolysis: 1. Phosphorylation of Glucose 2. Conversion of G-6-P to F-6-P 3. Phosphorylation of F-6-P to F-1,6-bisP 4. Cleavage of F-1,6-bisP 5. Interconversion of triose sugars 6. Oxidation of G-3-P to 1-3-bisPG 7. P Transfer from 1,3-bisPG to ADP 8. Conversion of 3-PG to 2-PG 9. Dehydration of 2-PG to PEP 10. Transfer of P from PEP to ADP

Answer 40

- Catalyst – hexokinase - Uses 1 ATP - ΔG = -16.7 kJ/mol – essentially irreversible NOTION 2.3

Answer 41

- Catalyst – phosphohexose isomerase - ΔG = 1.7 kJ/mol – proceeds either way due to low free energy NOTION 2.4

Answer 42

- Catalyst – phosphofructokinase-1 (PFK-1) - Uses 1 ATP - ΔG = -14.2 kJ/mol – essentially irreversible - 1st “committed” step of glycolysis, because G-6-P and F-6-P can be used in other pathways, but F-1,6-bisP is solely destined for glycolysis. NOTION 2.5

Answer 43

- Catalyst – fructose 1,6-bisphosphate aldolase (or aldolase for short) - ΔG = 23.8 kJ/mol – under cellular conditions the actual free energy change is small so the reaction is readily reversible - This is the “splitting” part of glycolysis. One Glc (6 C’s) is converted to two different 3C triose sugars NOTION 2.6

Answer 44

- Catalyst – triose phosphate isomerase - ΔG = 7.5 kJ/mol – low, so readily reversible reaction - Only G-3-P can participate in glycolysis, so the other 3 C sugar produced (dihydroxyacetone phosphate) is rapidly converted to G-3-P, thus yielding two G-3-P molecules for every one . NOTION 2.7

Answer 45

- Catalyst – glyceraldehyde 3-phosphate dehydrogenase - 2 NADH’s are produced - ΔG = 6.3 kJ/mol – endergonic, but see later - This is the first reaction in the “payoff” phase of glycolysis NOTION 2.8

Answer 46

- Enzyme – phosphoglyceratekinase - 2 ATP’s produced - ΔG = -18.5 kJ/mol – highly exergonic so spontaneous - Steps 6 and 7 are an energy-coupled process, so the overall ΔG is -12.2 kJ/mol - 1,3-bisPG is the reaction intermediate between this coupled process - This is one of the substrate-level phosphorylation reactions in glycolysis NOTION 2.9

Answer 47

- Catalyst – phosphoglycerate mutase - ΔG = 4.4 kJ/mol – in cells this is even lower, so reaction is reversible NOTION 2.10

Answer 48

- Catalyst – enolase - ΔG = 7.5 kJ/mol – again, in cells this is low, so reversible reaction can occur NOTION 2.11

Answer 49

- Catalyst – pyruvate kinase - 2 ATP’s produced - ΔG = -31.4 kJ/mol – highly exergonic, so reaction is spontaneous - This final step produces pyruvate NOTION 2.12

Answer 50

NOTION 2.13

Answer 51

NAD+ limited in the cell – comes from niacin (essential vitamin).

Answer 52

When an exercising muscle produces lactate from pyruvate, NADH is transformed back into NAD+. This fate, like many others, produces NAD+ to replenish the NAD+ required for reduction of various intermediate metabolites. This is known as “redox balance”.

Answer 53

It depends on what you need at the given time! The reactions that produce pyruvate from glucose are similar in most organisms. What happens to pyruvate next, is variable: - Ethanol - Lactate - CO2 NOTION 2.14

Answer 54

The 2 enzymes involved: - Pyruvate decarboxylase - Alcohol dehydrogenase NOTION 2.15

Answer 55

In human cells lacking O2 – Vigorously exercising muscle – RBC’s – lack mitochondria

Answer 56

Pyruvate is reduced to lactate via fermentation. Oxidation of NADH drives the reduction of pyruvate to lactate, which in turn replenishes stores of NAD+ for further glycolysis.

Answer 57

Enzyme = Lactate dehydrogenase NOTION 2.16

Answer 58

The Cori cycle is a metabolic process during which lactic acid produced in the muscles is converted to glucose by the liver and moved back to the muscles to be metabolized again.

Answer 59

- When we sprint, muscles don’t receive O2 fast enough to make ATP via oxidative phosphorylation - Instead ATP is made via substrate-level phosphorylation, producing lactate - This lactate would then enter the Cori Cycle

Answer 60

Lactate is converted to Glc in the liver by a process called gluconeogensis.

Answer 61

In cells with access to O2 the pyruvate is oxidised to form acetyl coenzyme A (acetyl CoA). This occurs within the mitochondria of cells. NADH formed in this reaction will later give up its hydride ion (:H-) to the respiratory chain.

Answer 62

NOTION 2.17

Answer 63

Some tissues which rely completely on glucose as their main energy source include: - The Brain - The Nervous System - Red Blood Cells - Testes - Embryonic Tissues

Answer 64

Just your brain needs 120 g of sugar a day out of the 160 g for the whole body.

Answer 65

The body has, - Approx. 20 g of free glucose in tissues - 190 g can be produced from glycogen stores - So, there’s enough to cover the 160 g daily requirement

Answer 66

7 out of 10 glycolysis reactions are reversible. Large –ve ΔG prevents the other 3 reactions being reversible.

Answer 67

Gluconeogensis is not a reverse of glycolysis. The cell bypasses the irreversible reactions with enzymes that catalyse a separate set of irreversible reactions. This causes glycolysis and gluconeogenesis to be irreversible processes.

Answer 68

• 4 reactions that sidestep the 3 irreversible reactions of glycolysis • This allows for independent control of the glycolysis and gluconeogenesis pathways • Also prevents them cancelling each other out • Utilise the cytosol (rxns C & D) and also the mitochondria (rxns A & B) NOTION 3.1

Answer 69

If pyruvate is substrate: Pyruvate (PYRUVATE CARBOXYLASE) Oxaloacetate ( (M) MALATE DEHYDROGENASE) Malate ( (C) MALATE DEHYDROGENASE) Oxaloacetate ( (C) PEP CARBOXYKINASE) PEP If lactate is substrate: Lactate (LACTATE DEHYDROGENASE) Pyruvate (PYRUVATE CARBOXYLASE) Oxaloacetate ( (M) PEP CARBOXYKINASE) PEP NOTION 3.2

Answer 70

- Like the glycolysis reaction (3) it is a control point for gluconeogenesis as it is irreversible under cellular conditions - If it were a direct reversal of the glycolysis reaction it would require phosphoryl group transfer from F-1,6-bisP to ADP, which is energetically unfavourable - Fructose 1,6-bisphosphatase catalyses the hydrolysis of fructose-1,6-bisphosphate -> Fructose-6-phosphate NOTION 3.3

Answer 71

- Third bypass reaction is the final step in gluconeogenesis - Dephosphorylation of G-6-P to glucose - Reversing the 1st glycolysis reaction would require phosphoryl group transfer from G-6-P to ADP, which is energetically unfavourable - Instead, this reaction is straight forward hydrolysis of the G-6-P - Glucose 6-phosphatase is the catalyst NOTION 3.4

Answer 72

• F-6-P is readily converted to G-6-P, which is usually the end point for gluconeogensis • Ending the pathway here allows the cell to “trap” the glucose – Remember earlier slides on “fates of glucose” • This final step to make free Glc takes place in the lumen of the ER NOTION 3.5

Answer 73

- Fructose and galactose can enter glycolysis at various points - Both fructose and galactose are common dietary carbohydrates - The body does not have pathways for catabolism of either of these sugars - Most fructose is metabolised by the liver

Answer 74

• Termed the fructose 1-phosphate pathway • Uses 1 or 2 ATP for each fructose molecule converted • 2 enzymes catalyse the conversion of two glycolysis intermediates – Fructose 1- phosphate aldolase – Triose kinase NOTION 3.6

Answer 75

• Galactose is converted to G-1-P through a sugarnucleotide derivative, UDP-galactose • UDP-glucose and UDPgalactose amounts remain unchanged as they are recycled, therefore the net product of this reaction is G-1-P NOTION 3.7

Answer 76

• Produces NADPH for all organisms - LIVER – fatty acid & steroid synthesis and drug metabolism - MAMMARY GLAND – fatty acid synthesis - ADRENAL CORTEX – steroid synthesis - RED BLOOD CELLS – as an antioxidant • Produces pentoses (5-C sugars) - These are precursors of ATP, RNA and DNA • Metabolises the small amount of pentose's in the diet - Usually dietary pentose’s come from digestion of nucleotides

Answer 77

P-P pathway has 2 phases: • Oxidative, irreversible part – Generates NADPH – Converts G-6-P to a pentose phosphate • Reversible, nonoxidative part – Interconverts G-6-P and pentose phosphate to form lots of different 3-, 4-, 5-, 6- and 7-C sugars NOTION 3.8

Answer 78

NADPH links catabolic and anabolic pathways. NOTION 3.9

Answer 79

• NADP+is used in exactly the same way as NAD+ - an electron carrier • NAD+ is used in metabolism of dietary sugars in the redox reactions of glycolysis and the citric acid cycle (see later lectures) • NADP+ is used in anabolism to convert simple precursors into things like fatty acids – NADP+ also acts as an antioxidant • Enzymes involved in both metabolic and anabolic pathways have differing specificities for these two electron carriers, which stops NADP+ being used for metabolism and vice versa

Answer 80

Ethanol -> Acetaldehyde -> Acetate -> Acetyl-CoA -> Stored as fat OR Citric Acid Cycle -> Terminal Respiratory System -> CO2 & H2O These steps use up a lot of NAD+! NOTION 3.10

Answer 81

So drinking inhibits gluconeogenesis • Leads to: – lacticacidaemia (increased [blood lactate]) – hypoglycaemia (decreased [blood Glc]) • And when untreated: – confusion → loss of consciousness → death! • Particularly bad if you’re: – athletic – dieting – a drunken bum

Answer 82

• G-6-P dehydrogenase deficiency: – Genetic condition affecting 400 million people worldwide • 1st step in the irreversible part of the P-P-P is catalysed by the enzyme G-6-P dehydrogenase • Symptoms under certain conditions: – Infection – Certain antibiotics – After eating fava beans (divicine) – Haemolytic anaemia – RBC’s burst, which darkens the urine with the iron they contain

Answer 83

• G-6-P dehydrogenase deficiency causes low RBC NADPH levels • This allows damaging free radicals and H2O2 to build up, which damages the RBC membranes • The damaged RBC are then unable to suffer the extra trauma of infections, divicine toxin etc.

Answer 84

• Prevalence of this condition suggests it confers an evolutionary advantage • As with sickle cell anaemia, carriers are resistant to the malaria parasite

Answer 85

NOTION 3.11

Answer 86

• PEPCK overexpression gives lots of PEP in muscle from lactate. • So, lactate → PEP → pyruvate (last step of glycolysis) • Pyruvate can enter the citric acid cycle (see next lectures) • Thus, lactate formed in the exercising muscles of PEPCKmus mice ends up in the citric acid cycle, which in turn provides energy for most of the ATP needed for muscle function • This way the mice can keep on going and going and going...

Answer 87

• Hans Krebs (1900 – 1981) • Found minced pigeon breast could use up “food” molecules without certain compounds in the muscle being used up – His idea was to propose a cyclical reaction – He won the 1953 Nobel Prize in Physiology and Medicine • Krebs, Hans, and Johnson, W. A. (1937). "The Role of Citric Acid in Intermediate Metabolism in Animal Tissues." Enzymologia4:148–156.

Answer 88

Citric Acid Cycle is also known as the Krebs Cycle, or Tricarboxylic acid (TCA) cycle.

Answer 89

It is the common metabolic pathway for all “fuel” molecules (carbohydrate, fatty acids and amino acids).

Answer 90

Occurs in the mitochondrial matrix

Answer 91

Unlike glycolysis, which yields a small amount of energy (just 2 ATP’s), this cyclic pathway yields much more energy that is then passed on to another biochemical system (the electron transport chain), which produces large amounts of ATP. It also manages to be part of catabolic processes. The cycle in collaboration with oxidative phosphorylation produces 90% of aerobic cell energy.

Answer 92

- No, the cycle does not produce ATP directly - It does not include O2 as a reactant - It removes e-’s and passes them on to form NADH and FADH2

Answer 93

It’s very efficient – Cyclical – Small number of citric acid cycle molecules can make loads of NADH and FADH2

Answer 94

• Primitive metabolism is believed to have been similar to glycolysis • Glycolysis (or something similar) yields no net oxidation of glucose (remember the NAD+/NADH redox recycling in glycolysis) • It therefore can’t yield all of the potential energy that glucose contains, so through fermentations you get small amounts of ATP (only 2 mol ATP per Glc molecule) • Thus, primitive organisms evolved ways of saving “some” of Glc’s potential energy

Answer 95

• The first plant-like organisms gave rise to increased atmospheric O2 • PROBLEM: O2 oxidises the very organic molecules that make up life • SOLUTION: become extinct! or survive by using up the abundant supply of O2 • Primitive organisms used this O2 to oxidise food molecules (e.g. Glc) further than was previously possible with the glycolysis reaction • This new use of O2 allowed the complete breakdown of high energy food molecules like Glc • These O2 using organisms therefore had an evolutionary advantage

Answer 96

Acetyl CoA sits in the centre of energy production for the cell as it allows different intermediates into the main energy producing pathway of the citric acid cycle. NOTION 4.1

Answer 97

• Acetyl CoA is synthesised from pyruvate, through the action of the enzyme pyruvate dehydrogenase. • Very complicated series of reactions involving decarboxylation of the pyruvate molecule, then oxidation, followed by transfer of the CoA complex • The decarboxylation step releases two electrons (in the form of 2 H ions), which can pass to O2 to produce more ATP through NADH intermediates NOTION 4.2

Answer 98

NOTION 4.3

Answer 99

It’s MASSIVE – 50 nm across. It contains tens of copies of each enzyme sub-unit (E1, E2, E3) Each sub-unit catalyses a different part of the reaction to convert pyruvate to acetyl CoA - E1catalyses the first decarboxylation of pyruvate - E2transfers the acetyl group to coenzyme A - E3 recycles the lipoyllysine through the reduction of FAD, which is recycled by passing electrons to NAD+ NOTION 4.4

Answer 100

NOTION 4.5

Answer 101

Steps in CAC: - Oxidation of Acetyl CoA to CO2 and H2O - Highlighted C’s are from Acetyl CoA - CoA is used again in step 4 then removed again in step 5 - Alpha-ketoglutarate dehydrogenase reaction is similar to pyruvate dehydrogenase NOTION 4.6

Answer 102

Breaths per day: 15 x 60 x 24 = 21600 breaths Inhaled O2 per day: 0.2 x 500 x 21600 =2160000 ml CO2 produced: 2160000 x 0.25 = 540000 ml

Answer 103

Molecules: 0.05 x 540 x 6.022 x 10^23 = 1.62594 x 10^26

Answer 104

• Pyruvate dehydrogenase is regulated by it’s immediate products and the end point of cellular respiration, ATP • It’s regulated depending on the needs of the cell: NOTION 4.7

Answer 105

Both points of control are at non-reversible reactions (exergonic steps). The first one = isocitrate dehydrogenase The second one = Alpha-ketoglutarate dehydrogenase

Answer 106

Isocitrate dehydrogenase: - As with pyruvate dehydrogenase, this enzyme is allosterically controlled through ATP and NADH concentrations - ATP and NADH will negatively regulate - ADP positive regulates

Answer 107

α-ketoglutarate dehydrogenase: - Again, ATP and NADH negatively regulate - Also, succinyl CoA negatively regulates

Answer 108

– Blocking isocitrate dehydrogenase causes citrate build up (isocitrate and citrate are interconvertible), which shuttles citrate into the cytoplasm causing phosphofructokinase to stop glycolysis – α-ketoglutarate builds up when α-ketoglutarate dehydrogenase is inactive, which switches it’s use to production of amino acids

Answer 109

Citric Acid Cycle is an amphibolic pathway, as it serves both catabolic and anabolic processes. NOTION 4.8

Answer 110

• When cellular energy needs are met through the citric acid cycle, it can produce the building blocks of nucleotide bases, heme groups and proteins • One problem: this depletes the cell of citric acid cycle intermediates • In exercising muscle the cells require ATP, which depletes the amount of oxaloacetate • Remember back to gluconeogenesis, pyruvate can be converted to oxalocaetate by the enzyme pyruvate carboxylase • Pyruvate carboxylase is only active when acetyl CoA is present, so a build up of acetly CoA triggers this reaction • This is known as an anaplerotic reaction (Greek origin, meaning to “fill up”)

Answer 111

NOTION 5.1

Answer 112

NOTION 5.2

Answer 113

The mitochondria

Answer 114

Allows the coupling of the oxidation of carbon fuels to ATP synthesis. Utilises proton gradients to produce ATP (and lots of it).

Answer 115

• The majority of NADH and FADH2 is formed in the mitochondrial matrix), but some NADH is formed in the cytoplasm (gycolysis) • A shuttle is used to move reducing equivalents across the mitochondrial membrane • Cytoplasmic NADH cannot cross the membrane, but FADH2 can pass its e-’s on to the electron transport chain within the mitochondria • This process is termed the glycerol phosphate shuttle

Answer 116

• The NADH is unable to cross the membranes of the mitochondria, but G-3-P can, passing its e-’s to FADH2 • Oxidation of FADH2 in the electron transport chain generates, per mol, less ATP than oxidation of NADH • Thus, an energetic ‘price’ is paid for using cytosolic reduced cosubstrates in terminal respiration NOTION 5.3

Answer 117

NOTION 5.4

Answer 118

Complex I: NADHQ oxidoreductase • Oxidises NADH and passes the high-energy e-’s to ubiquinone to give ubiquinol (QH2) • Utilises Fe-S centres and FMN (flavin mononuleotide) • Pumps H+ ions into the intermembrane space NOTION 5.5

Answer 119

Amytal (barbiturate), piercidin A (antibiotic) and rotenone (insecticide) all inhibit eflow from Fe-S centres to ubiquinone and therefore block the electron transport chain.

Answer 120

Complex II: Succinate-Q reductase • Oxidises FADH2 and like complex I passes high-energy e-’s to ubiquinone, which becomes ubiquinol (QH2) • Utilises Fe-S centres to channel e-’s • This enzyme is also part of the citric acid cycle under the guise of succinate dehydrogenase

Answer 121

Heme group is believed to block stray e-’s. If they “leak” from the system it is believed they can contribute to cancer by forming free radicals of O2 (superoxide). Point mutations in this protein near the heme group give rise to hereditary paraganglioma (benign tumours in the head and neck – particulalry in the carotid body – an organ that senses blood O2).

Answer 122

• Called Q10 in mitochondria (as it has 10 isoprene repeats) • CoenzymeQ10 is its other name • Dietary supplement believed to reduce free radicals and thus act as an antioxidant.

Answer 123

Complex III: Qcytochrome c oxidoreductase • Takes the e-’s from ubiquinol (QH2) and passes them to cytochrome c • 1 QH2 is oxidised to yield two reduced cytochrome c molecules • Pumps protons into the intermembranespace NOTION 5.6

Answer 124

Complex IV: Cytochrome c oxidase • Takes the e-’s from cytochrome c and passes them to molecular O2 • e-’s channeled through Fe-Cu centre • Pumps protons into the intermembrane space NOTION 5.7

Answer 125

• Energy is conserved from the breakdown of food molecules and ultimately leads to the oxidation of NADH, FADH2, ubiquinone and cytochrome c • Energy is further conserved through the setting up of a proton gradient across the inner mitochondrial membrane. NOTION 5.8

Answer 126

• ANSWER (part 1): remember the electron motive force in Bioenergetics lectures – in this case it is a proton motive force – this allows the gradient to do work • ANSWER (part 2): a molecular turbine has evolved to harness the energy in the proton gradient – ATP synthase

Answer 127

It is both: - Chemical potential because the inside is alkaline - Electrical potential because the inside is negative

Answer 128

As e-’s pass through the complexes of the transport chain protons move from the matrix to the outside of the inner mitochondrial membrane – chemiosmosis. These reactions have particular spatial directionality, so are classed as ‘vectoral’. This is an example of an energy transformation.

Answer 129

• Protons eventually flow down their concentration gradient, back into the matrix of the mitochondria • Only a relatively small number of sites exist on the membrane were this happens • At these sites exists a large multi-unit protein complex called ATP synthase (ATPase for short) • ATPase has a central pore that allows protons to pass through • As they flow through the pore the energy stored in the gradient is used to convert ADP + Pi to ATP

Answer 130

ATP Synthase has 2 parts: - F0: Membrane bound proton conducting unit - 10 subunits - Separate subunit connects F0 to F1 - F1: Protrudes into the mitochondrial matrix and acts as the catalyst for ATP synthesis - Produces ATP from proton motive force

Answer 131

• ADP + Pi enters β subunit • Rotation of F0 cylinder and γ shaft forces conformational changes in the β subunits of F1 • Conformational changes catalyse the ADP → ATP conversion and release ATP NOTION 6.1

Answer 132

Reaction coordinate diagram shows the proton gradient drives release of ATP and not formation of ATP. NOTION 6.2

Answer 133

• Protons move from positive side of membrane (H+P) to the negative side (H+N) • Sequential conformational changes of β subunit: – β subunit that binds ADP + Pi – β subunit that binds ATP – β subunit that doesn’t bind ATP NOTION 6.3

Answer 134

• In vitro analysis of the F1 portion of ATP synthase with the central γ subunit can show the mechanism of rotation • Actin filament has fluorescent compounds incorporated into it so the movement can be visualised NOTION 6.4

Answer 135

Stated previously (glycerol phosphate shuttle slide) that oxidation of FADH2 in the terminal respiration system generates less ATP than NADH oxidation.

Answer 136

As e-’s pass through the electron transport chain, complexes I, III and IV move a total of 8 H+from the matrix to the outside of the membrane.

Answer 137

ATP synthase can produce one ATP for every 3 H+ it moves back into the matrix across the membrane.

Answer 138

• NADH feeds in e-’s at Complex I, which means the e-’s pass through all 3 sites in the chain that allow protons to move across the membrane (Complexes I, III and IV) • FADH2 feeds in at Complex II, so only two of the sites that move protons across the membrane are utilised (Complexes III and IV)

Answer 139

Stoichiometrically, approximately 2.5 and 1.5 mol of ATP are generated per mol of NADH and FADH2 respectively.

Answer 140

NOTION 6.5

Answer 141

Electron transport is said to be coupled to ATP synthesis.

Answer 142

• If the inner mitochondrial membrane becomes permeable to protons, the proton gradient cannot be generated • If this happens the electron transport can still occur, with O2 being reduced to H2O, but no ATP is made • The two processes are now uncoupled

Answer 143

The energy released from e-’s passing along the terminal respiration system does not make ATP, it is released as heat.

Answer 144

Malignant hyperthermia is a disease caused by ‘leaky’ mitochondrial membranes that uncouple electron transport and ATP synthase.

Answer 145

- Susceptible people, exposed to halothane (or halothane-like drugs) experience a large increase in their body temperature. - Halothane is believed to make the inner mitochondrial membranes in muscle leaky in some way - Muscle cells will usually become irreversibly damaged from the excessive heat build up.

Answer 146

1 in 75,000 people (1:15,000 children)

Answer 147

A similar thing (to malignant hyperthermia) happens in pigs: – Porcine stress syndrome – When shipped to market they can become ‘stressed’ following halothane exposure – Results in pork meat that is watery and very low pH (i.e., pickled)

Answer 148

Intentional uncoupling • Brown fat in newborn infants • Also in other mammal species • Brown fat cells have lots of mitochondria • If a baby becomes cold, norepinephrine triggers the opening of a channel in a protein called thermogenin • Thermogenin sits on the inner mitochondrial membrane of brown fat cells

Answer 149

Intentional uncoupling • Even plants can do it! • Arum lily to attract insects • Skunk cabbage to melt the snow that covers it • Similar mechanism to mammals used to uncouple oxidative phosphorylation to generate heat