1
Q
Summarise the five main functions of the Krebs cycle.
A
- metabolic engine
- oxidation of Acetyl-coA
- CO₂ emission
- reducing equivalent production (i.e. NADH and FADH₂)
- GTP production
(approx. 10 ATP/cycle produced)
2
Q
State the reducing equivalents and how they react in the Krebs cycle.
A
- NAD⁺ reduction as it loses a hydride (H-) ion and a proton (H⁺): NAD⁺ → NADH + H⁺
- FAD⁺ undergoes sequential reduction by hydrogen addition: FAD⁺ + H⁺ → FADH + H⁺ → FADH₂
3
Q
Describe how enzymes of the Krebs cycle are named.
A
- oxidoreductase → oxidation reactions to produce NADH and FADH₂
- dehydrogenases → reactions involve loss of hydrogen (H)
4
Q
Describe reaction 1 of the Krebs cycle.
Hint - Owen and Chris 2- the enzyme synthesizes this product
A
- favourable under standard conditions
- reaction driven by formation of citryl-coA intermediate which undergoes rapid hydrolysis
- oxaloacetate + acetyl CoA → citrate (citrate synthase)
- alongside: H₂O → CoA
(draw out using notes)
5
Q
Describe reaction 2 of the Krebs cycle (2-step isomerisation).
(Hint - Can’t Carry It 2)
A
- step 1: citrate -H₂O → cis-acotinate/intermediate (aconintase)
- step 2: cis-acotinate/intermediate +H₂O → isocitrate (aconintase)
- coordination of OH group in step 1
- facilitation of rehydration in step 2 by active site 4Fe-4S cluster
- mitochondrial form of aconitase enzyme used
6
Q
Describe reaction 3 of the Krebs cycle (2-step decarboxylation and formation of NADH).
(Hint - India Owns A 3)
A
- step 1: oxidation:
isocitrate → (isocitrate dehydrogenase) oxalosuccinate intermediate - Step 2: decarboxylation
oxalosuccinate intermediate → (isocitrate dehydrogenase) α-ketoglutarate - isocitrate dehydrogenase tightly-regulated
- metabolic nodal point – other pathways produce α-ketoglutarate
7
Q
Describe reaction 4 of the Krebs cycle (oxidative decarboxylation with NADH formation)
(Hint - Andy 1-2-3 Splat 4)
A
- enzymatic mechanism with 3 subunits which catalyse different stages of reaction
- α-ketoglutarate → [E1 +TPP (+ ↪CO₂) → E2 + lipoamide (+ ↪CoA) → E3 + FAD⁺ (+ ↪ NAD⁺ → NADH + H⁺)] aka the α-ketoglutarate dehydrogenase complex (intermediate) → succinyl CoA
- note 1. is decarboxylation and 3. is oxidation of lipoamide
(see notes for details)
8
Q
Describe reaction 5 of the Krebs cycle (substrate-phosphorylation).
(Hint - Sally Surfs 5)
A
- succinyl-CoA → Succinate (succinyl-CoA synthase)
- alongside: GDP + ADP → (nuceloside diphosphate kinase) GTP + ATP
- enters E.T.C
(phosphoryl transfer)
9
Q
Describe reaction 6 of the Krebs cycle (reduction)
Hint - Sianise Flies 6
A
- succinate → (succinate dehydrogenase) fumarate
- alongside reduction: FAD → FADH₂
- FADH₂ enters E.T.C via reduction of coenzyme Q10 in inner membrane of mitochondria
- succinate dehydrogenase associated with membrane, and covalently-linked to FADH₂ molecule
10
Q
Describe reaction 7 of the Krebs cycle (hydration)
Hint - Freya Can’t Match 7
A
- fumarate → (fumarase + OH-) carboanion intermediate (fumarase + H⁺) → malate
- hydration of C=C catalysed by stereospecific enzyme
- formation of L-isomer of malate which is important for metabolite transporters across mitochondrial membranes
11
Q
Describe reaction 8 of the Krebs cycle (oxidation)
Hint - Marley Owns 8
A
- malate → oxaloacetate (malate dehydrogenase)
- alongside: NAD⁺ → NADH + H⁺ (reduction)
- oxidation of OH group regenerates oxaloacetate
12
Q
How is the Krebs/Citric acid cycle studied and what happens to the carbons from acetyl CoA and how are some carbons lost?
A
- carbons from Acetyl-coA are not ones lost via decarboxylation reactions
- acetyl-coA carbons become incorporated into oxaloacetate at the end of the cycle
- thus lost as CO₂ in subsequent turns of cycle
- explored using 14C-labelling experiments
(see notes for summary drawing of cycle)
13
Q
What drives the Krebs cycle?
Hint - to do with ΔG and certain steps
A
- ΔG difficult to determine as in mitochondria and isolating w/o contaminating with cytosol difficult
- steps 1, 3 and 4 → key regulatory points
- step 8 not spontaneous with low oxaloacetate concentrations → can drive reaction forward
14
Q
Name five enzymes which are key in regulating the Krebs cycle.
(Hint - PICAP - 1x creation, 3x remove H, remove CO₂ - α)
A
- citrate synthase
- isocitrate dehydrogenase
- α-ketoglutarate dehydrogenase
- pyruvate dehydrogenase
- pyruvate decarboxylase
15
Q
Describe oxidative phosphorylation in mitochondria.
A
- 4 enzymes complexes (lots of proteins in them) embedded in membrane
- NADH as e- donor passes through complex 1 (ubiquinone/nol) and 4 H⁺ pass through
- with FADH₂ less ⁺ pumped → less ATP synthesis
• QH is shuttle molecule
• terminal complex so oxygen reduced to water
(see notes for diagrams)
16
Q
What is reduction potential and what does it mean?
A
- tendency of a species to accept electrons/become reduced
- more positive the value the more likely to accept electrons
- relative to a proton
- biochemical standard reduction potential (E°’) is for a compound under standard conditions (like G°’)
- as you go along ETC reduction potential of substrates increases
- O₂ + 2H⁺ + 2e- → H₂O
17
Q
What is the general formula of saturated fatty acids?
A
- CH₃(CH₂)nCOOH
- contain HC chain, terminal carboxylic acid group and cis or trans bonds
18
Q
Give 4 examples of saturated fatty acids.
A
- stearic acid; octadecanoic acid, 1st C=C at C1, 18 Cs
- oleic acid; ω 9-fatty acid, 1st C=C at C9, 18 Cs
- linoleic acid; ω 6-fatty acid, 1st C=C at C6, 18 Cs
- α-linoleic acid; ω 3-fatty acid 1st C=C at C3, 10 Cs
(see notes for details)
19
Q
Describe triacylglycerol metabolism.
A
- glucose can be converted into fatty acyl glycerol
- hormone-sensitive lipase used to conjugate fats to lipids and then transport fats
- intracellular (unlike from lipoprotein + pancreatic lipase)
- TAG → DAG → MAG → FFA + glycerol
TAG = triacyl glycerol DAG = diacyl glycerol MAG = monoacyl glycerol FFA = free fatty acid
20
Q
What do high glucose levels cause for hormone-sensitive lipase?
A
- inhibition of lipolysis (fat breakdown)
- provides glycerol-3-phosphate from glycolysis
- thus, promotes fat storage
21
Q
What are the 4 main roles of fatty acids?
Hint - Faisah Served Colourful Halvah
A
- fuel molecules – mostly stored in adipose tissue as triacylglycerols (i.e. triacylglycerol metabolism)
- structural – component of lipid membranes as attached to groups
- covalent modifications of proteins (dynamic and reversible)
- increase interaction w/ membranes (lipid anchors)
- promote protein-protein interactions - production of fatty acid hormones - i.e. prostaglandins stimulate inflammation, modulate synaptic transmission, stimulate sleep
22
Q
Why is fatty acid synthesis regulated?
A
- so catabolism (mitochondria) and anabolism (cytoplasm) don’t simultaneously occur (pathways not simply reversals of one another)
23
Q
What is stage 1 of fatty acid synthesis?
Hint - AM and lots of other molecules involved
A
- acetyl CoA → (acetyl CoA carboxylase, ACC) malonyl CoA
- alongside: HCO₃- + ATP → ADP + Pi
- first step, irreversible, rate-limiting, allosteric enzyme
- requires biotin (vitamin B7) and ATP
(see notes for details)
24
Q
What is stage 2 of fatty acid synthesis?
Hint - CoACP
A
1) acetyl CoA → acetyl ACP (acetyl trancylase)
- alongside: ACP → CoA
2) malonyl CoA → malonylACp (malonyl transacylase)
- alongside: (another) ACP → CoA
- ACP is a transport protein which binds to acetyl
25
What are the final stages of fatty acid synthesis?
elongation steps:
- feedback into addition of 2C from condensation w/ malonyl-ACP
- condensation, reduction, dehydration, reduction...
(see notes for diagram)
26
Describe acetyl-coA availability for fatty acid synthesis.
- shuttle pathway used to move acetyl CoA from mitochondria to cytosol
- NADPH produced and used
27
Describe regulation of acetyl Co-A carboxylase (ACC) activity by insulin and glucagon.
(Hint - all things CIG)
- citrate used to partially switch on fat breakdown pathway (partially actives carboxylase)
- allosteric (citrate) and hormonal (insulin + glucagon) regulation of lipolysis
- insulin → activates ACC
- glucagon → deactivates ACC
(see notes for diagram)
28
Describe fatty acid oxidation (activation).
| Hint - +A or -P
- adenosine added to activate fatty acyl-CoA
- molecule then sent into mitochondria
- pyrophosphate + fatty acyl-adenylate → fatty acid
- removal of pyrophosphate helps thermodynamically drive reaction forward
29
Why is beta-oxidation needed and what type of process is it?
- to transport activated fatty acids across membranes
- (inner fatty acids more impermeable to membrane)
- a mitochondrial process
30
State the 4 stages of beta-oxidation.
| Hint - IHOT
1. intro of a trans C=C bond between α and β Cs
- FADH₂ produced (enters respiratory chain as e- donor)
- 3 forms of mitochondrial Acyl-CoA dehydrogenase (short C4-8, medium C8-14 and long C12-18 chain FAs)
- associated w/ inner membrane of mitochondria – feeds electrons into Complex II
2. hydration of trans C=C (introduced in 1st reaction) to introduce C=O group on beta carbon atom
3. another oxidation – at beta C to produce ketone (O=C-C-C=O) group instead of OH
- formation of NADH
- involves the enzyme 3-Hydroxyacyl-CoA dehydrogenase
4. thioloysis
- lysis of α-β C-C bond
- addition of a coenzyme A moiety (group) to β carbon
- production of acetyl-CoA
- acyl-CoA produced can re-enter oxidation cycle
31
What are the equations for the 4 stages of β-oxidation?
| Hint - FeaTuring 3 different βAs
1. fatty acyl CoA → trans-Δ₂-enoyl-CoA (acyl CoA dehydrogenase)
- alongside: FAD → FADH₂
- intro of a trans C=C bond betw/ α and β carbons
- mitochondria have 3 versions of Acyl-CoA dehydrogenase (short, medium and long chain fatty acids)
2. trans-Δ₂-enoyl-CoA → 3-L-Hydroxyacyl-CoA (enoyl-CoA hydratase)
- alongside: hydration (+H₂O) of trans C=C → C=O
3. 3-L-Hydroxyacyl-CoA → β-ketoacyl-CoA (3-hydroxyacyl-CoA dehydrogenase )
- alongside: NAD⁺ + H⁺ → NAD⁺
- oxidation to produce ketone (O=C-C-C=O) from C=O
4. β-ketoacyl-CoA + CoA (moiety) → acyl-CoA + acetyl-CoA ( β-ketoacyl-CoA thiolase)
- lysis of α-β C-C bond
- produced Acyl-CoA which can re-enter oxidation cycle
- then move into Complex III
32
Give a summary of numbers of net products for the oxidation of long-chain, even-numbered, saturated fatty acids (e.g. palmitate).
```
• beta oxidation:
- 7x NADH → 17.5x ATP
- 7x FADH₂ → 10.5x ATP
- 8x acetyl CoA
↓
• citric acid cycle:
- 24xNADH → 60x ATP
- 8x FADH₂ → 12x ATP
- 8x GTP → 8x ATP
• Total = 108x ATP per palmitoyl CoA
(see notes for diagram)
```
33
What is ketogenesis?
| Hint - ABAK
- conversion: acetoacetate + β-hydroxybutyrate → acetyl-CoA → ketone bodies
- cannot feed into citric acid cycle + drive ATP production
34
Where is ketogenesis usually seen?
| Hint - when there is some sort of problem
- disease states of defective glucose metabolism/starvation (liver cells)
- → lack of glucose metabolism
- → oxaloacetate pyruvate (gluconeogenesis)
- → reduced flux of CAC + acetyl-CoA accumulation
- → excess acetyl-CoA provides energy in peripheral tissues (skeletal and cardiac muscle)
35
What normally occurs with β-hydroxybutyrate in the body?
| Hint - 2 conversions and then cut into CoAs and used for CACs
- → (oxidised) to acetoacetate + succinyl-coA → acetoacetyl-CoA
- next step cannot occur in liver cells (lack enzyme)
- → (cleavage) acetoacetyl-CoA → 2x acetyl-coA → entry to citric acid cycle (mitochondria)
36
How is ketogenesis caused and what are its symptoms and implications?
(Hint - high levels of 2 certain ketones, what this leads to; symptoms - Mikky Ekko song, getting crazy, GI pain)
- too high levels of beta-hydroxybutyrate + acetoacetate in the blood can cause a drop in blood pH
- occurs when more is produced than can be used by peripheral tissues
- symptoms; nausea, vomiting, stomach pains, high acetone levels (fruity breath odour), delirium, comatose
(there are cases where diabetics with ketoacidosis have died)
37
There are a range of different enzymes which work on overlapping chain lengths of fatty acyl-CoA. What is MCADD and its symptoms and treatment?
- medium-chain acyl-CoA dehydrogenase deficiency (MCADD)
- inability to break down fat as an energy source
- severity of enzyme deficiency is variable
- symptoms: hypoglycaemia, build-up of medium length fatty acids, fatigue, seizures
- treatment: feeding glucose regularly, enzyme replacement therapy
N&E
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