Lecture 19: The TCA Cycle Flashcards
1
Q
What is the citric acid cycle?
A
Shitty way to word the question, but…
- More ATP generated from glucose than glycolysis
- Aerobic conditions
- Take place in mitochondria vs cytosol
2
Q
What is acetyl CoA?
A
- Acetyl coenzyme A
- Carbon source (via acetyl group) for citric acid cycle
- Generated from pyruvate before entry into cycle (decarboxylated)
3
Q
Explain the stuff (sorry…) about how the citric acid cycle occurs in the mitochondrial matrix
A
- power centers of the cell
- membrane-bound organelles with a double membrane
- outer membrane is fairly smooth
- inner membrane is highly convoluted, forming folds called cristae
- cristae greatly increase the inner membrane’s surface area
- Location of ATP production
4
Q
Give an overview of the products of oxidation?
A
- 2 CO2 molecules
- 1 GTP
- 8 Electrons in the form of NADH and FADH2
- See Slide 6 and get ready to piss yourself
5
Q
Describe cellular respiration (I know, i know. These setups are garbage)
A
- High energy electrons are removed from carbon fuels
- Electrons reduce O2 - generating a proton gradient (red)
- Gradient used to synthesize ATP (green) in oxidative phosphorylation
6
Q
Describe the relationship between glycolysis and the TCA cycle
A
- Carbohydrates—glucose—can be converted to pyruvate in glycolysis
- Pyruvate from glycolysis can be converted to acetly CoA
- Under anaerobic conditions pyruvate is converted to lactate or ethanol
- Aerobic conditions result in pyruvate entering the mitochondria
- See Slide 9
7
Q
What is pyruvate dehydrogenase?
A
First step is to convert pyruvate to acetyl CoA
…among other things.
8
Q
How is acetyl CoA produced from pyruvate
A
- Requires 3 enzymes and 5 coenzymes
- Catalytic cofactors
- Thiamine pyrophosphate (TPP)
- Lipoic acid
- FAD
- Stoichiometric cofactors
- CoA
- NAD+
- 3 steps:
- Decarboxylation
- Oxidation
- Transfer acetly group to coenzyme A
- Reactions are coupled to preserve energy to drive formation of NADH and acetyl CoA.
- See Slide 12 for reaction equation. And the book for having ANY hope of doing well on this next test.
9
Q
Describe decarboxylation
A
- Pyruvate combines with TPP is then decarboxylated to hydroxyethyl-TPP
- Catalyzed by pyruvate dehydrogenase component E1 using TPP as the prosthetic group
- H of TPP is very acidic(Loss of Hydrogen Proton)
- Carbanion readily attacks carbonyl group of pyruvate(Gain Hydrogen Proton) and decarboxylation of pyurvate occurs(CO2 Product) before protonation(Gain of Hydrogen Proton)
10
Q
Describe Oxidation:
A
- Hydroxyethyl group attached to TPP is oxidized to an acetly group
- Acetyl group is transferred to lipoamide (lipoic acid derivative) linked to a lysine residue of E1
- Creates a energy rich thioetser bond in acetyllipoamide
11
Q
Describe the formation of acetyl CoA
A
- Acetly group transferred to CoA
- E2 catalyzes reaction
- Energy rich thioester bond remains
- Acetyl CoA used in citric acid cycle
12
Q
Describe pyruvate dehydrogenase regeneration
A
- Dihydrolipoamide must be reconverted to lipoamide to conduct another reaction
- Oxidized form of lipoamide regenerated by E3
- 2 electrons transferred to FAD, then to NAD+ (unique)
- Electron transfer potential of FAD increased because it is tightly associated with the enzyme
13
Q
Describe the pyruvate dehydrogenase complex
A
- 24 copies of E1
- 24 copies of E2
- 12 copies of E3
- Each E2 subunit is a trimer containing 3 distinct domains
- Flexible arm of E2 containing lipomide allows all domains of the complex to work together
14
Q
Describe the first step of the pyruvate dehydrogenase reaction
A
- Pyruvate decarboxylation at E1
- Hydroxyethyl-TPP intermediated formed
- CO2 is first product
15
Q
Describe the second step of the pyruvate dehydrogenase reaction
A
- Lipoamide arm of E2 is inserted into a deep E1 channel containing the active site
16
Q
Describe the third step of the pyruvate dehydrogenase reaction
A
E1 catalyzes transfer of acetyl group to lipomide chain -acetylated arm enters E2 active site
17
Q
Describe the fourth step of the pyruvate dehydrogenase reaction
A
- Acetly moiety transferred to CoA and second product, acetyl CoA leaves
- reduced lipomide arm swings to active site of E3 FAD containing protein
18
Q
Describe the fifth step of the pyruvate dehydrogenase reaction
A
- At active site E3, lipomide is oxidized by coenzyme FAD
- reactive lipomide ready for another cycle
19
Q
Describe the sixth step in the pyruvate dehydrogenase reaction
A
- Final product, NADH is produced with reoxidation of FADH2 to FAD
20
Q
Describe how the proximity of the enzymes effects the pyruvate dehydrogenase reaction
A
Proximity of all enzymes increases reaction rate and keeps all intermediates bound and are readily transferred as the flexible arm of E2 calls on each active site in turn
21
Q
What are the three enzymes and 5 coenzymes needed in the process of
A
- Requires 3 enzymes
- E1
- E2
- E3
- 5 coenzymes
- Catalytic cofactors
- Thiamine pyrophosphate (TPP)
- Lipoic acid
- FAD
- Stoichiometric cofactors
- CoA
- NAD+
22
Q
Describe pyruvate dehydrogenase regulation
A
- Regulated allosterically and by reverse phosphorylation
- High [acetyl CoA] directly inhibits E2
- Products also increase phosphorylation of PDH E2, while accumulation of ADP and pyruvate activate phosphatases
23
Q
Describe the key points
A
- The citric acid cycle occurs under aerobic conditions and produces more energy from glucose than glycolysis
- The citric acid cycle takes place in the mitochondria
- Pyruvate dehydrogenase (PDH) links glycolysis to the citric acid cycle
- PDH contains 3 enzymes and uses 5 cofactors to generate acetyl CoA for entry into the citric acid cycle
- See Slide 30-31
24
Q
Describe what happens when the Citric acid cycle oxidizes 2-carbon units
A
- First step is condensation of 4-carbon oxaloacetate and 2-carbon acetly group of acetly CoA
- Oxaloacetate reacts with acetyl CoA + water yields citrate and CoA
- Catalyzed by citrate synthase
- See Slide 33
25
Describe how Citrate isomerizes to isocitrate
- Hydroxl group of citrate is not in proper location for oxidative decarboxylation
- Dehydration/hydration moves OH atoms via the enzyme aconitase (uses iron-sulfur cluster to bind citrate)
- See Slide 35
26
Describe Isocitrate Dehydrogenase
* First of 4 oxidation-reduction reactions
* Unstable intermediate, oxalosuccinate, loses CO2 while bound to the enzyme
* This step is important in determining the rate of the cycle
- Allosterically stimulated by ADP, enhancing enzyme affinity for substrate
* Also Where an NAD+ is converted to an NADH
- See Slide 37
27
Describe alpha ketoglutarate dehydrogenase
* Complex similar to pyruvate dehydrogenase
* Catalyzes similar reaction
* Both reactions decarboxylate an α-ketoacid and create a thioester linkage with CoA
- NAD+ and CoA go in; NADH, CO2, and H+ come out
- See Slide 39
28
Describe succinyl CoA synthetase
- Succinyl CoA contains a high energy thioester bond (ΔG°' similar to ATP)
- Only step that directly yields a high energy phophotransfer compound (GTP)
- See Slide 41
29
Describe the 4-carbon compound reactions
* Final stage of cycle
* Regeneration of oxaloacetate
* Methylene group (CH2) is converted to carbonyl group (C=O) via 3 steps
* Methylene group (CH2) is converted to carbonyl group (C=O) via 3 steps
- Oxidation
- Hydration
- Oxidation
* Oxaloacetate is regenerated for another round of the cycle and more energy is extracted to form FADH2 and NADH
30
Describe Succinate dehydrogenase with regards to ATP production
* Succinate dehydrogenase catalyzes formation of fumarate while generating FADH2
* The enzyme is located in the inner mitochondrial membrane directly associated with electron transport chain
* FADH2 is actually not released from the enzyme, but electrons are passed directly to coenzyme Q in the electron transport chain
- See Slide 45
31
Describe Malate Dehydrogenase
* Final step
* Oxidation of malate has a positive standard free energy
* Therefore, reaction is driven by the use of the products
* Oxaloacetate - citrate synthase
* NADH - electron transport chain
- See Slide 47 & 48
32
Describe Citric Acid Cycle Regulation (...huge)
* TCA cycle is important source of energy and building blocks for many important biomolecules
- Pyruvate can be converted to glucose and used in glycolysis, but pyruvate to acetly CoA is irreversible
- Either TCA cycle or lipids
* Entry into the cycle and the rate of the cycle are controlled at several stages
* Regulated allosterically and by reverse phosphorylation
* High [acetyl CoA] directly inhibits E2
* Products also increase phosphorylation of PDH E2, while accumulation of ADP and pyruvate activate phosphatases
- Energy charge of cell dictates PDH activity
* Products also increase phosphorylation of PDH E2, while accumulation of ADP and pyruvate activate phosphatases
* These phosphatases are also stimulated by Ca2+ which increases to initiate muscle contraction
- Providing energy where needed
* Also, insulin can stimulate fatty acid synthesis by activating phosphatases and increasing the conversion of pyruvate to acetyl CoA (precursor for fatty acids)
* Controlled at many points
* First is isocitrate dehydrogenase
* Allosterically stimulated by ADP, enhancing enzyme affinity for substrate
* Reaction product, NADH also inhibits by directly displacing NAD+
Second control site is α-ketogluturate dehydrogenase
* Remember: this complex is similar to PDH
* Also similar to PDH in regulation
- Allosterically inhibited by its products, succinyl CoA and NADH
* Control at these sites help integrate the TCA cycle with other pathways
* Ex. Control at isocitrate dehydrogenase leads to build up of citrate (easily converted from isocitrate) which can transport to the cytosol and signal phosphofructokinase and halt glycolysis
* Also, α-ketoglutarate that builds up from enzyme inhibition can be used for synthesis of amino acids and purine bases
33
Describe Citric Acid Cycle Intermediates
- When energy needs are met, intermediates are drawn for biosynthesis of other molecules
- They are replenished by formation of oxaloacetate from pyruvate
34
Describe Pathway Integration
Pathways active during exercise after a night’s rest
| - See Slide 58
35
Describe Anaplerotic Reactions
* Branch chain amino acids are a source for citric acid cycle intermediates
1. Amino acids converted to pyruvate Alanine, serine, glycine, threonine, cysteine, tryptophan
2. Amino acids converted to oxaloacetate Aspartate, asparagine
3. Amino acids converted to αketoglutarate Glutamate, glutamine, proline, histidine, arginine
4. Amino acids converted to fumarate Phenylalanine, tyrosine
5. Amino acids converted to succinyl-CoA Methionine, isoleucine, valine
6. Amino acids converted to acetyl-CoA Leucine, isoleucine, lysine, phenylalanine, tyrosine, tryptophan, threonine
36
Describe the Key Points of Anaplerotic Reactions ( I think)
- The citric acid cycle oxidizes 2 carbon units
- Entry to the citric acid cycle and metabolism through it are highly controlled
- PDH is regulated both allosterically and by phosphorylation
- The citric acid cycle is a source of biosynthetic precursors
- Anaplerotic reactions are required during states of low energy
