Final Flashcards
(124 cards)
1
Q
The regulation of glycolysis happens at the______step of glycolysis -
A
irreversible; working to control the levels of ATP and pyruvate within the cell
2
Q
_____ of proteins, fats and proteins in the 3 phases of cellular respiration; ____ oxidises stuff into ___
A
Catabolism; Kreb’s cycle; CO2
3
Q
The pyruvate Dehydrogenase complex (PDC)
A
The PDC catalysed reaction occurs in the mitochondrial matrix
Enzyme bridges glycolysis to the Krebs cycle
The product of glycolysis Pyruvate cannot go directly into the Krebs cycle-> must convert pyruvate into Acetyl coA
This reaction involves a decarboxylation/oxidation of pyruvate in the form of a thioester, followed by the formation of acetyl CoA
Trap energy of oxidation in the thioester -> can use thioester to do work later on
4
Q
Dehydrogenase
A
NAD+ will be involved and a redox reaction is occurring/ being oxidized
5
Q
The pyruvate Dehydrogenase complex (PDC)
COMPOSITION:
A
PDC is composed of 3 enzymes and 5 cofactors Cofactors: Thiamine pyrophosphate (TTP) - bound to E1 Lipoamide - bound to E2 NAD+ - free/not bound FAD (oxidized) - bound to E3 CoASH - free Enzymes: E1: pyruvate dehydrogenase (Differentiate on exam if you are talking about PDC or pyruvate dehydrogenase) E2: dihydrolipoyl transacetylase E3: dihydrolipoyl dehydrogenase
6
Q
Coenzyme A (aka CoA or CoASH) + reaction
A
Structure of CoASH and acetyl coA below
CoASH = empty, nothing bound but thiol
Composed of ADP, pantothenate (vitamin B5) and B-mercaptoethylamine
Carrier of acyl groups
Attaches to acyl/carboxyl groups with hydrocarbon chain
Forms high energy thioester bonds
AcetylCoA + H2O ⇌ acetate + CoASH ; △G°’: -31kJ/mol (ATP is sound -30)
7
Q
Thiamine pyrophosphate (TPP)
A
derived from vitamin B1 (thymine) and it forms a reactive carbanion easily
carries aldehydes
8
Q
Lipoic acid/lipomide:
A
Lipoic acid is attached to a lysine in E2 is called lipoamide
Has a disulfide group that can be oxidised or reduced
Acts like a an robotic am: oxidise aldehydes into acyl group, resulting in the acyl group being bound via the disulfide group
Can move things from active site to active site
9
Q
Mechanism of pyruvate dehydrogenase complex:
A
- Pyruvate enter E1 m binds to TPP and is decarboxylated to form the intermediate hydroxyethyl-TPP
- The oxidised lipoamide arm enters E1
- The hydroxyethyl group is oxidised to an acetyl group and bound to the lipoamide arm
Note: the lipoamide arm has been reduced to a dihydrolipoyl group (reduced lipoamide arm) - The arm (carrying the acetyl group) moves into E2 and the acetyl group is transferred to CoASH, forming acetyl CoA
Acetyl coA leaves the enzyme, forming main product - The reduced lipoamide arm moves into E3 where it is oxidised by FAD. FAD is reduced to FADH2
- NAD+ enters E3 and reoxidises FADH2 back to FAD. NAD+ is reduced to NADH + H+ which now leaves E3 -> now back to step 1
10
Q
Mechanism of pyruvate dehydrogenase complex
- extra info
A
This reaction happens over and over again
This reaction connects glycolysis to the Kreb’s cycle
This reaction is Heavily controlled - regulation of the pyruvate dehydrogenase complex
11
Q
regulation of the pyruvate dehydrogenase complex
A
- High [acetyl CoA] allosterically inhibit E2 - where acetyl coA is made
- High [NADH] allosterically inhibit E3
- The MAIN CONTROL is at E1 where there is a kinase associated with PDC (PDC associated kinase)
When PDC associated kinase is active, it phosphorylates E1, causing E1 to slow and thus the entire complex to slow down - Acetyl coA and NADH (i.e. products) all stimulate the PDC associated kinase
Activation of the kinase -> inhibition of the enzyme - Buildup of pyruvate and NAD+
- There are general phosphatases that will gradually dephosphorylate E1, returning it to its regular state
- There is a PDC associated phosphatase that when activated by cell signalling (such as increase in [Ca2+] and insulin will rapidly dephosphorylate E1
12
Q
regulation of the pyruvate dehydrogenase complex
Buildup of pyruvate and NAD+
A
Pyruvate, NAD+ (reactants), and ADP all inhibit the kinase to not slow down the complex (activate enzyme)
More ADP means less ATP, and more NAD+ means less NADH, and more pyruvate means less glucose -> need more energy so inhibit kinase to stop the slowing down (i.e. speed up) of pyruvate dehydrogenase complex
Leading to more ATP and energy production
Inhibition of kinase -> allows the enzyme to function
13
Q
regulation of the pyruvate dehydrogenase complex
There are general phosphatases that will gradually dephosphorylate E1, returning it to its regular state
A
Gradually over time, dephosphorylate to E1 -> if you do not have a constant phosphorylation signal, you will restore the enzyme to its higher active state
14
Q
regulation of the pyruvate dehydrogenase complex
There is a PDC associated phosphatase that when activated by cell signalling (such as increase in [Ca2+] and insulin will rapidly dephosphorylate E1
A
There are signals that can lead to very rapid dephosphorylation of the PDC
Insulin is the hormone that gives permission to burn glucose -> not surprising that insulin would activate PDC -> to go ahead an oxidise the sugar
15
Q
Kreb’s cycle (aka citric acid cycle and the tricarboxylic acid cycle, TCA)
A
Main job is to oxidise things -> to generate create high energy electrons to be used in oxidative phosphorylation to make ATP
The krebs cycle is the Central hub of metabolism of the cell
The krebs cycle Completely oxidises acetyl coA to CO2 and in the process generates high energy e- (in the form of NADH and FADH2) and GTP
These e- can be used in oxidative phosphorylation to generate ATP
the krebs cycle is also a source for many biological precursors (makes things)
Occurs in the matrix of the mitochondria
Stuff has to go in to be pulled out of the krebs cycle -> if just pulled out without putting in, it will destroy Kreb’s cycle
Start with oxaloacetate and must be regenerated as it is a cycle
16
Q
Kreb’s cycle
Reaction 1
(DRAW NOW)
A
loading the molecule in reaction
Citrate synthase forms citrate by binding oxaloacetate to acetyl CoA
Going from c4 to c6
Aldol condensation to form citryl coA
Attach acetyl CoA to oxaloacetate
Hydrolysis of citryl coA to form citrate and coASH
Negative △G°’
Resonance
Coupled with hydrolysis (cleaving) of Thioester
Citrate is quite symmetrical
17
Q
Kreb’s cycle
Reaction 2
(DRAW NOW)
A
reposition the OH
Aconitase converts citrate to isocitrate
Moving/repositioning the OH group
Dehydration reaction to form cis-aconitate and induce double bond, followed by a hydration step to generate isocitrate
△G°’ is positive but the reaction is driven forward by reaction 1 & 3, the concentration of products + reactants
Note: the OH is moved on to the CH2 that originated as oxaloacetate not from acetyl coA
Because C2 has pseudo chirality, the enzyme can distinguish between methylene from C1 and C3
18
Q
Kreb’s cycle
Reaction 3
(DRAW NOW)
A
Isocitrate is oxidised and then decarboxylated to alpha-ketoglutarate by isocitrate dehydrogenase
Electron carrier needed (NAD+)
NADH & CO2 are produced
Isocitrate is oxidised to oxalosuccinate, generating NADH
Oxalusuccinate is decarboxylated (spontaneously) to alpha-ketoglutarate
5 carbon
Note: technically the CO2 lost did not originate from the acetyl coA that just entered to the cycle
Negative △G°’; not happy compound
19
Q
Kreb’s cycle
Reaction 4
(DRAW NOW)
A
Alpha-ketoglutarate is decarboxylated/oxidised and bound to coASH (thioester formation) by the alpha-ketoglutarate dehydrogenase complex, generating succinyl coA, CO2, and NADH
Occurs by the same method as pyruvate dehydrogenase complex
I.e. same cofactors, similar E2 and E1 and identical E3 enzymes
Back to 4 carbons
Negative △G°’
20
Q
Kreb’s cycle
Reaction 5
(DRAW NOW)
A
Slightly Negative △G°’(Coupling thioester hydrolysis to GTP production)
Succinyl coA synthetase converts succinyl coA to succinate, generating GTP & coASH
Named in backwards direction (reversible reaction) can make succinyl coA if GTP is used
The reaction is driven by the negative △G of the cleavage of the thioester bond
Note: GTP can be converted to ATP by a nucleoside diphosphate kinase
GTP + ADP ⇌ GDP + ATP
This happens all the time
Note: there are isoforms of succinyl coA synthetase that use ADP
The next steps are involved in the regeneration of oxaloacetate from succinate
Succinate is completely symmetrical
Oxaloacetate is the carboxylated form of pyruvate
21
Q
Kreb’s cycle
Reaction 6
(DRAW NOW)
A
Succinate dehydrogenase oxidises succinate generating FADH2 and fumarate (trans)
Free energy change is not high enough to reduce NAD+
Succinate dehydrogenase is part of complex II (part of electron transport chain)
22
Q
Kreb’s cycle
Reaction 7
(DRAW NOW)
A
Fumarase adds water across the double bond, forming L-malate
We are adding an OH group
23
Q
Kreb’s cycle
Reaction 8
(DRAW NOW)
A
Malate dehydrogenase oxidises L-malate to oxaloacetate, generating NADH
Cycle is complete - the Krebs cycle is the main supplier of electrons to the electron chain
24
Q
Synthase
A
an enzyme catalysing a synthetic reaction in which 2 unit are joined without the direct participation of ATP
Citrate is product when using citrate synthase
25
Synthetase
an enzyme catalysing a synthetic reaction in which 2 unit are joined with the direct participation of ATP (required)
26
Overall (net) equation of the Krebs cycle
Acetyl CoA (Main fuel) + 3NAD+ + FAD + GDP + Pi + H2O -> 2CO2 + 3 NADH + 2H+ + FADH2 + GTP + CoASH
To generate high energy electrons -> goal of krebs cycle
Water is needed for fumarase reaction and water is needed to cleave of CoA
Isocitrate dehydrogenase reaction, it required an H+ to remove the CO2 -> so there is only 2H+ in product instead of 3H+
1 molecules of glucose -> 2 turns of the krebs cycle
27
Regulation of the cycle
1. Isocitrate dehydrogenase
2. Alpha-ketoglutarate dehydrogenase complex
3. citrate synthase (optional - only occurs in bacteria)
Generally ATP and NADH slows the cycle down
28
Isocitrate dehydrogenase
Stimulated allosterically by ADP
| ATP and NADH inhibit allosterically the isocitrate dehydrogenase
29
Alpha-ketoglutarate dehydrogenase complex
Inhibited allosterically by NADH, ATP, succinyl coA
| Succinyl coA is the product from Alpha-ketoglutarate; the product is allosterically inhibited the substrate
30
citrate synthase
(optional - only occurs in bacteria)
Inhibited allosterically by ATP
Hopeful to be a good antibiotic to knockout krebs cycle of bacteria
They tried but it was toxic to other parts of our bodies
31
Oxidative phosphorylation
the formation of ATP as a result of the transfer of e- from NADH and FADH2 to O2 by e- carriers
32
Oxidative phosphorylation
| electron motive force, EMF
The e- attached to NADH and FADH2 have high transfer potential (aka the electron motive force, EMF) (chemical gradient)
EMF can be harnessed by the electron transport chain (ETC) to transfer protons out of the mitochondrial matrix, through the inner mitochondrial membrane (IMM) and into the intermembrane space (IMS)
33
Oxidative phosphorylation
| proton motor force (PMF)
The resulting electrochemical gradient forms a proton motor force (PMF) (electric gradient)
This PMF can be used to by ATP synthase to generate mechanical spin and generate ATP (a molecule with high phosphoryl transfer potential)
34
Note about Mitochondria
The ETC and ATP synthases are embedded in the inner mitochondrial membrane (IMM)
Cristae -> surface area to fit ETC and ATP synthase
IMM is packed full of ETC and ATP synthase
IMM is impermeable to small molecules and ions -> very good barrier
IMM requires transporters/transport proteins to move things across it
The outer mitochondrial membrane (OMM) is porous and permeable to small molecules and ions
OMM is considered leaky because it has many pores and this the IMS is similar to cytosol
(and is often referred to as the cytosol)
35
Electron transfer and thermodynamics
| E- can be transferred as:
```
1. H: hydride ion
E.g. NADH
2. H: hydrogen atom
E.g. FADH2
3. Free e-
Eg. ETC - jump from different molecules
```
36
standard reductive potential (Eo’)
Different molecules have different tendencies to accept e-
This can be measured as standard reductive potential (Eo’) in volts
The more positive the Eo’, the higher the molecules affinity for e-
Measured in electrochemicals using hydrogen electrode at pH 7 as standard
Eo’ of oxygen is 0.82 volts -> has a high affinity for electrons and so oxygen is used as the final electron acceptor
37
NAD’s affinity for electrons (-0.320) is a lot less than oxygen
(overall reaction of the ETC)
NAD+ + 2e- + 2H+ -> NADH + H+ (Eo’: -0.320)
½ O2 + 2e- + 2H+ -> H2O (Eo’: 0.820)
I.e. O2 has a higher affinity for e- than NAD+
Conversely, NADH is more likely to donate e- than H2O
The 2 half reactions must be coupled in order for e- to be transferred
NADH + H+ -> NAD+ + 2e- + 2H+
½ O2 + 2e- + 2H+ -> H2O
____________________________
NADH + ½ O2 + H+ -> NAD+ + H2O
38
△G°’ can be related to Eo’ by the following:
△G°’ = -nF△Eo’
Where
n = # of e-
F = Faraday’s constant 96.5 kJ/Vmol
For the 2 half reactions forming a redox reaction
△Eo’ = Eo’(e- acceptor) - Eo’(e- donor)
Note: these values are taken directly from the table - no sign flipping (because (-) already has the flip)
Must use this equation for the final equation
39
E- transfer from NADH to O2
△G°’ = -nF△Eo’
△Eo’ = Eo’(O2) - Eo’(NADH)
= 0.82 V - (-0.32 V)
= 1.14 V
Now calculate △G°’
△G°’ = -2(96.5kJ/Vmole)(1.14 V)
= -220 kJ/mole
-220 kJ/mol (e- transfer from NADH to O2)
Divided by 30.5 kJ/mol (ATP synthesis) = ~7
In theory, if we used every last Joule, we could make 7 ATP
But in reality, some energy will be lost as heat and oxidative phosphorylation
so we will generate ~2.5 ATP/NADH
Cytosolic NADH must be moved into the mitochondrial matrix
40
The Electron Transport Chain
E- are transferred through a series of e- carriers (most of which are embedded in complexes I - IV) of increasing △Eo’ until they reach O2, the final e- acceptor
in the process H+ are moved into IMS
Each carrier as we move along the chain has a higher affinity for electrons than the carrier before it
Goal is create a proton gradient
The ETC is composed of 4 major complexes, each containing multiple proteins and e- carriers
There are also 2 electron carriers that act as shuttles, moving electrons from complex to complex
41
Complex I: NADH - Q oxidoreductase
NADH - Q oxidoreductase; onramp to the ETC for matrix NADH
Accepts 2e- from NADH (NADH on ramp)
Proton pump
Electrons are transferred to FMN, and then a series of 4Fe-4S clusters
And then finally to coenzyme Q (ubiquinone) reducing it to QH2 (ubiquinol)
If you moves these 2 e- through the entire complex, this results in 4H+ being pumped out of the matrix and into the IMS
Net equation for complex I:
NADH(matrix) + 5H+(matrix) + Q -> NAD+(matrix) + QH2 + 4H+(IMS)
Total 6 protons are moving around
2 of which (1 from NADH and one proton from the matrix) will make QH2
The remaining 4 protons go into the IMS contributing to the proton motor force (proton gradient)
42
Complex II: succinate-Q-(oxido)reductase
Succinate dehydrogenase is part of this enzyme
The electrons from succinate -> fumarate are transferred to FAD (forming FADH2) then to Fe-S clusters in the succinate-Q reductase, and then finally to Q forming QH2
I.e. these are the electrons from the Krebs cycle FADH2
Complex II is how e- from FADH2 enter the krebs cycle
Using these Fe-S clusters because they typically have optimal negative △Eo’
As we get more to oxygen, we will need positive △Eo’ so we cannot always use Fe-S clusters
Complex II is not a H+ pump: No protons being pumped here
The △G is negative but not negative enough to pump protons
Electrons from FADH2 do not move as many protons as NADH across the IMM
Note: e- from NADH do NOT pass through complex II
Heme B is an electron carrier
43
Heme B is an electron carrier
has a very positive (+) △Eo’
Backup system to prevent the release of uncarried electrons
Complex I and II are different onramps to the ETC
44
Coenzyme Q
(ubiquinone/ubiquinol) acts as a shuttle, moving e- from complex I and II and others to complex 3
Small hydrophobic molecule located in the IMM
Contains a repeating isoprenoid tail
# of repeats varies from species to species (Q10: humans have 10 repeats)
Ubiquinone can accept 2e- and 2 protons to be reduced to ubiquinol
Q + 2e- + 2H+ ⇌ QH2
Q is free to move around only in the IMM but IMM is so full of complexes, Q cannot move around much
45
Complex III: Q-cytochrome c oxidoreductase
Contains:
2Fe-2S cluster
2 cytochromes
Cytochrome b heme bL & bH
Cytochrome c1 heme c1
Take the 2e- from QH2 (oxidising it back to Q) and thansfers them one at a time to the 2Fe-2S clusters them to heme c1 and finally to heme C in cytochrome C
Occurs via the Q cycle
Net equation for complex III:
QH2 + 2Cyt c(oxidised) + 2H+ (mat) -> Q(oxidised) + 2Cyt c(reduced) + 4H+(IMM)
Complex III pumps protons (main goal) and gets e- from QH2 and puts them onto C
46
Cytochrome
e- transferring protein containing one or more heme groups
47
Q cycle
the process of transferring e- from ubiquinol (QH2) to cytochrome C
QH2 comes in and 1 e- moves up to the Fe-S cluster to Heme c1 and to Heme C onto cytochrome C and cytochrome c will roll away
While 1e- is moving up the system, the other e- goes to heme b and waits until the first e- is out of the way after which the other e- moves up to the Fe-S cluster to heme c1 then to Heme C and cytochrome c the system
cytochrome b (with its hemes) is used to move an e- into a holding pattern and waits
In the possess (assuming 2e- move through complex III), 4H+ are moved into the IMS
2H+ come directly from the matrix
2H+ come from the QH2
Note these protons come from the matrix in one of the other complexes
48
Cytochrome c
| and where is it
Another e- shuttle
Contains heme c
Water soluble protein containing a covalently linked heme
Carries 1e- from complex III to complex IV
Cytochrome c likes to be around the intermembrane space side of the IMM (sits on the surface of IMM) - rolls along the surface of the membrane
Fe3+ + e- -> Fe2
49
Complex IV: Cytochrome c Oxidase
| + composition
Proton pump
Carries out final reduction of oxygen to water using e- from Cyt c
End of electron transport chain
Note: To fully reduce O2 to 2H20 requires 4 electrons; in the process, 4H+ are moved into the IMS
Complex IV contains:
2 cytochromes
Cyt a -> heme a
Cyt a3 -> heme a3
2 copper centres
CuA
CuB
Heme a3 and CuB form one key centre as they are so close together
The O2 binds to Heme a3 and bridges between Heme a3 & CuB
e- flow is from heme c to CuA to heme a and then finally to Heme a3/CuB centre
Net reaction from complex IV:
2 Cyt c(reduced) + 4H+ (mat) + ½ O2 -> 2Cyt c(oxidised) + H2O + 4H+(IMS)
Complex IV is designed to prevent the release of partially reduced O2
Complex IV does not care the cyt c comes from as long as it gets it
50
Complex IV: Cytochrome c Oxidase
| e.g. ROS
O2- (superoxide; O2 with an unpaired electron) & O2-2 (peroxide)
These are known as reactive oxygen species (ROS)
ROS can damage DNA & proteins
51
Some molecules can bind to the various e- carriers in the ETC and block e- transfer
for example N3- (azide), CO, CN- (cyanide) can all bind the iron in Heme a3 in complex IV
Prevents e- flow to O2, and thus, blocks ETC, preventing formation of the proton gradient and block ATP synthesis
52
ATP Synthase and ATP synthesis
We need to harness the H+ product created by the ETC to generate ATP which is done by ATP synthase
pH difference is 1.4 units; 40 times more protons in IMS than IMM
ATP synthases is composed of 2 components
Fo is embedded in the IMM and contains half channels that the protons flow through
It uses H+ flow to create spin
F1 extends into the matrix and synthesises ATP when coupled to spin from Fo
53
F1
contains 3𝞪𝜷 subunits arranged in a ball (ATP synthesis occurs in 𝜷) in the centre of the ball is the 𝞬 (gamma) shaft
The 𝞬 shaft is asymmetric (each of the 3 sides has unique shape) and binds to each 𝞪𝜷 subunit differently, causing different conformations in each of the 𝞪𝜷 subunits
results in 3 different conformations
Using the H+ gradient, Fo causes the 𝞬 shaft to spin!
Only the gamma shaft is spinning not the 𝞪𝜷 ball
load -> make -> release (repeat)
This causes each of the 3 𝞪𝜷 subunits to cycle through the loose, tight, and open conformations
Result: generation and release of ATP
54
F1 results in 3 different conformations
1. 𝞪𝜷 open conformation: low affinity for ATP & ADP; i.e. this is the release conformation
(O; 𝜷-empty)
2. 𝞪𝜷 loose conformation: ADP and Pi can bind and become trapped. I.e. this is the loading conformation
(L; 𝜷-ADP)
3. 𝞪𝜷 tight conformation: ATP is generated but is tightly bound to 𝜷
(T; 𝜷-ATP)
55
Best estimates suggest _ protons are needed to make 1 ATP molecule
4 (3 to spin Fo and 1 to move Pi)
56
PO values
NADH: (10 H+/NADH)/(4H+/ATP) = 2.5 ATP/NADH (PO value [phosphate oxygen])
FADH2: (6H+/FADH2)/(4H+/ATP) = 1.5 ATP/FADH2
I.e. These are # of ATP 2e- can generate in oxidative phosphorylation
Phosphate: oxygen values
57
How does Fo cause the 𝞬 shaft to spin?
Fo is composed of many subunits but focus on two, subunit c and subunit a
Subunit c is composed of 2 𝞪-helices that span the membrane
There are 10-12 c subunits arranged in a cylinder
Halfway down one of the 𝞪-helices is a key aspartate/aspartic acid which can be protonated or deprotonated depending on pH
Deprotonated in the matrix side
The entire c subunit cylinder will spin
58
The subunit a (aka the clamp)
Subunit a covers 2 c subunits
Has 2 half channels (they only run halfway down the membrane)
One open is open to IMS where the aspartate is
One open to the matrix in the middle where the aspartate is
Subunit a is stationary
59
Subunit c can only move into (i.e. be exposed to) the membrane if it is ...
... uncharged (i.e. aspartic acid), but it can move when charged if it is covered by subunit a (subunit a “masks” the charge)
I.e. subunit c can exist as an aspartate as long as it is covered by subunit a, which is hiding the charge from the hydrophobic membrane
But it cannot go into the membrane unless it is an aspartic acid and the charge is gone
60
How does proton (H+) flow cause Fo to spin the c cylinder?
1. A charged aspartate subunit c is in the IMS half channel.
A charged aspartate subunit c is in the matrix half channel
2. A H+ diffuses from the IMS through the IMS half channel and protonates the aspartate to an uncharged aspartic acid
3. The c cylinder complex can rotate clockwise by one c subunit (note it can’t rotate counter clockwise). The newly uncharged aspartic acid c subunit enters the membrane
4. This brings a charged aspartate c subunit into the IMS half channel and a fresh aspartic acid c subunit into the matrix half channel
5. A proton diffuses off the aspartic acid, down the matrix half channel and into the matrix. The c subunit is now a charged aspartate
Back to step 1
61
controlled brownian motion
The motion of ATP synthase is believed to be derived from controlled brownian motion (acts like motor)
Note: a proton from the IMS binds a charged aspartate subunit c and then goes almost 1 full rotation of the cylinder before being released into the matrix
Minimum 8/10 subunits must be protonated for the ATP synthase to spin
Does not rotate smoothly because it builds up torque but the release and accepting the substrates
Can change speed by changing actin filament length and shape (longer the actin, the slower the spin)
62
Regulation of oxidative Phosphorylation
Normally ATP synthesis and the ETC are coupled (the rates are linked)
The proton gradient couples them
The ETC makes the proton gradient and the ETC uses the proton gradient
ATP is only formed as fast as it is consumed
When the ratio of [ATP] / [ADP][Pi] is high, there is little ADP to be phosphorylated, O2 consumption drops
When [ATP] / [ADP][Pi] is low, reverse -> known as acceptor control
Acceptor control: the regulation of cellular respiration by the availability of ADP as a phosphate acceptor
Note: ATP synthase can only spin if ADP and Pi are bound in the loose conformation of the 𝞪𝜷
ATP synthase will not spin unless loaded with ADP
63
If ATP levels are low, we want to make more ATP and there is a lot of [ADP]
The spin of ATP increases
The protons are speeding up
For a split nanosecond, the proton gradient begins to drop -> becomes easier to pump protons into the IMS
O2 consumption speeds up and ETC speeds up and NADH levels drop as it is being used up
NAD+ levels climb as NADH is being used up
The Krebs cycle then speeds up as NAD+ levels climb
Therefore, ADP levels controls the whole system
The availability of ADP to be phosphorylated controls the whole process of cellular metabolism, cellular respiration, etc
64
2,4 dinitrophenol
2,4 dinitrophenol can uncouple the ETC and ATP synthase by carrying protons across the IMM, reducing the proton gradient
Dinitrophenol likes to live in membranes and has an OH group that is balanced such that it will protonate if it comes against the IMS side and deprotonate if it comes across the matrix side
ETC speeds up but ATP synthase remains the same (or if 2,4 DNP is really high ATP synthase dops) and heat is produced
Not using all the energy to make ATP; converting fuel stores into heat
No way to turn off when drug is in the system
Was used as a diet drug, but since it produces heat, people would die by cooking themselves by frying their heart and lungs (failed diet drug) and develop cataracts (temperature messed with their eyes)
65
Thermogenin
Brown fat carries Thermogenin (uncoupling protein 1) found in newborns and in hibernating animals
Thermogenin is a proton (H+) channel in the inner mitochondrial membrane (IMM) -> way to generate heat
We lose this ability in adulthood -> downregulate expression of thermogenin
Controllable system - can control how much thermogenin is made
Allow newborn baby or hibernating animal to generate heat from their fat preserves - reason why hibernating animals do not freeze to death
66
Transport across the IMM (good barrier)
Electron transport to the ETC
Q: Specifically how do the electrons on cytosolic NADH get to the ETC?
NADH normally cannot cross membranes
Especially from glycolysis
A: occurs via shuttle system: we use one of 2 shuttle systems to move electrons to the ETC
1. Glycerol-3-phosphate shuttle (skeletal muscle, brain)
2. malate-aspartate shuttle (liver, kidney, heart)
67
Transport across the IMM (good barrier)
| ADP, ATP and Pi
ATP and ADP are transported by the ATP/ADP translocase
ATP/ADP translocase is next to ATP synthase
Exchanges 1 ATP in the matrix for 1 ADP in the cytosol
Driven by the charge gradient created by the proton motor force
ATP has a charge of -4 and ADP has a charge of -3 and the cytosol is positively charged with respect to the matrix
Pi is transported by the phosphate translocase
This pumps 1 Pi and 1 H+ into the matrix
Driven by the PMF
68
Transport across the IMM (good barrier)
| Pyruvate
Pyruvate is moved into the matrix by pyruvate translocase
69
Glycerol-3-phosphate shuttle (skeletal muscle, brain)
1. Cytosolic glycerol-3-phosphate dehydrogenase reduces DHAP (dihydroxyacetone) to glycerol-3-phosphate. In the process, NADH is reoxidized to NAD+
Glycerol-3-phosphate is carrying 2e- to the IMS
2. IMM bound glycerol-3-phosphate dehydrogenase reoxidizes glycerol-3-phosphate back to DHAP. DHAP goes back to the cytosol.
FAD is reduced to FADH2
3. FADH2 passes the e- to Q (becoming FAD again), reducing Q to QH2. QH2 can go to complex III
Note: this is similar to complex II
e- bypass complex I by entering as FADH2 and you use the FADH2 PO value
NADH is worth 1.5
70
Does the Glycerol-3-phosphate shuttle system screw up glycolysis?
No because we simply regenerate DHAP that has been consumed
| As long as the concentration of DHAP is maintained, it will not screw up the system
71
malate-aspartate shuttle (liver, kidney, heart)
1. Oxaloacetate in the cytosol is reduced by NADH to malate. The NADH is oxidised back to NAD+ (can go back to let glycolysis to continue)
2. Malate (carrying the 2e-) is transported into the matrix by the malate/alpha-ketoglutarate translocase
3. Malate is oxidised back to oxaloacetate, reducing mitochondrial matrix NAD+ to NADH -> This NADH can go to complex I
However, we have a problem: oxaloacetate cannot be directly transported back to the cytosol
(there is no oxaloacetate translocase)
4. Oxaloacetate is transaminated (moving an amino group) by glutamate, forming aspartate and alpha-ketoglutarate in the matrix
By ripping off amino group of glutamate, it turns into alpha-ketoglutarate
Amino group attached to oxaloacetate it turns into aspartate
5. Aspartate and alpha-ketoglutarate are transported into the cytosol
6. In the reverse reaction of step 4: aspartate transaminates alpha-ketoglutarate, regenerating oxaloacetate and glutamate
Like krebs cycle but backwards
NADH is worth 2.5 as normal
72
Total ATP yield for the complete oxidation of glucose to CO2 & H2O
from 1 molecule of glucose
Glycolysis
2 NADH
| 2 ATP
73
Total ATP yield for the complete oxidation of glucose to CO2 & H2O
from 1 molecule of glucose
Pyruvate dehydrogenase complex
2 NADH
| Matrix - does not need shuttle system
74
Total ATP yield for the complete oxidation of glucose to CO2 & H2O
from 1 molecule of glucose
Krebs cycle
```
(2 turns to break down glucose)
matrix - does not need shuttle system
6 NADH
2 FADH2
2 ATP (GTP)
```
75
Total ATP yield for the complete oxidation of glucose to CO2 & H2O
from 1 molecule of glucose
Oxidative phosphorylation
NADH from glycolysis
NADH from glycolysis
2 x 2.5 (assuming using aspartate malate shuttle)
```
5 ATP (3; if using glycerol-3-phosphate shuttle - FADH2 shuttle so times 1.5 instead of 2.5)
Can pick which shuttle system you want to use if not indicated
```
76
Total ATP yield for the complete oxidation of glucose to CO2 & H2O
from 1 molecule of glucose
Oxidative phosphorylation
NADH from PDC
2 x 2.5
| 5 ATP
77
Total ATP yield for the complete oxidation of glucose to CO2 & H2O
from 1 molecule of glucose
Oxidative phosphorylation
NADH from Krebs
15 ATP
78
Total ATP yield for the complete oxidation of glucose to CO2 & H2O
from 1 molecule of glucose
Oxidative phosphorylation
FADH2 from Krebs
3 ATP
79
Oxidative phosphorylation gives __ ATP
Oxidative phosphorylation gives 32 ATP (30)
| - Final exam question: calculate ATP equivalent yield for particular molecule
80
Glycogen metabolism
Maintaining blood glucose levels are critical for human life
81
There are 2 ways for body to restore low blood glucose in the body:
(liver does both)
1. Glycogen breakdown: Liver stores of glycogen and breakdown to produce glucose
2. Gluconeogenesis (making glucose from scratch
82
Glycogen
polymer of glucose molecules -> used to store glucose
83
Why do we need to store glucose as glycogen? (3)
1. brain uses almost exclusively glucose (or ketone bodies)
Glycogen allows for the release of glucose when blood glucose is low
2. Glucose from glycogen can generate ATP without O2 -> glycolysis
3. Glycogen can be broken down very quickly in times of high activity
84
There are 2 tissue types that stores high levels of glycogen
Liver tissue -> for body wide use via the bloodstream (mostly the brain)- does not operate for own use
Keto diet - we are tricking the liver and forcing it to do gluconeogenesis
Skeletal muscle tissue -> for muscle use only
85
Glycogen metabolism
| Liver
Structure: glycogen is stored as large granules in the cytosol
Glycogen granules consists of highly branched/rods of polymers of glucose -> created a lot of ends which is useful for breakdown
Each chain of glucose consists of 12-14 glucose residues connected by alpha(1->4) linkages
At around the 5th or 6th residue, there is a branch point where two chains are linked by an alpha(1->6) linkage
Each chain usually has 2 branch points
86
The glycogen chain has polarity
Reducing end: free C1 anomeric carbon- there is only 1 or maybe 2 in the whole glycogen polymer in the centre (the rest are attached by branch points)
Nonreducing ends: the many other ends have free C4OH
87
Glycogen synthesis
| step 1
Glucose-6-phosphate (from hexokinase in glycolysis) is converted to glucose-1-phosphate
By Phosphoglucomutase -> moves phosphate group around
88
Glycogen synthesis
| step 2
An activated form of glucose is made by attaching glucose to uridine triphosphate (UTP), forming uridine diphosphate glucose (UDP-glucose) and pyrophosphate
Catalysed by UDP-glucose pyrophosphatase
Technically this reaction is reversible but pyrophosphate is rapidly hydrolyzed to 2 orthophosphate (Pi) by pyrophosphatase
This reaction is irreversible and therefore makes the synthesis of UDP-glucose pretty much irreversible
89
Glycogen synthesis
| step 3
Glycogen synthase transfers glucose from UDP-glucose to the non-reducing end of a growing glycogen outer branch chain, forming a new alpha(1->4) linkage
This results in glycogen(n+1) & UDP
Note: the synthesis of glycogen costs energy
Glycogen synthase required a primer of at least 4 linked glucose residues
This is initially supplied by the enzyme glycogenin
90
Glycogen synthesis
| step 4
Branched chains are formed by branching enzyme
Transfers a 7 glucose residue segment from the non-reducing ends of an outer chain of at least 11 residues and forms an alpha(1->6) linkage initially to the same chain or to a neighbouring chain
This makes many non-reducing ends and makes glycogen more soluble
If you do not branch (which humans must do), you would have starch
91
Glycogen Breakdown
| step 1
Glycogen phosphorylation removes a glucose residue from the nonreducing-end of an outer chain of glycon by the addition of inorganic phosphate
Known as phosphorolysis
Delta G is just negative enough to move reaction forward in certain conditions
Yields glucose-1-phosphate and glycogen(n+1)
Note: glucose is phosphorylated without the use of ATP
Glycogen phosphorylase stops 4 terminal residues before a branch point
92
Glycogen Breakdown
| step 2
Debranching occurs via 2 steps
A transferase (from debranching enzyme) shifts 3 residues from one branch to the other
The remaining alpha(1->6) linkage glucose is then hydrolyzed (i.e. using water) by alpha-1,6-glucosidase and yields free glucose (not glucose-1-phosphate)
Alpha-1,6-glucosidase is also a part of debranching enzyme
93
Glycogen Breakdown
| step 3
Phosphoglucomutase converts glucose-1-phosphate into glucose-6-phosphate
94
Fate of glucose-6-phosphate (3)
1. Glycolysis (muscle- fight or flight mode)
2. Dephosphorylation by glucose-6-phosphatase (hydrolyzes the phosphate off) and release into the bloodstream (liver - restore blood glucose levels and brought into brain)
3. Pentose phosphate pathway to generate ribose and NADPH (in most cells) in liver
95
Gluconeogenesis
the process of generating glucose from non-carbohydrate precursors (making glucose from scratch)
Occurs in liver (and a little bit in the kidney) to maintain blood glucose levels
96
Molecules that can be used to make glucose
1. lactate/pyruvate
2. Protein/AA
3. glycerol
97
Molecules that can be used to make glucose
| lactate/pyruvate
Lactate is the reduced form of pyruvate
During times of high muscle activity glucose is fermented into lactate
What happens to lactate? (2 options)
- Option 1: cori cycle:
- Option 2: the heart can take up lactate
98
Molecules that can be used to make glucose
| Protein/AA
Either from the diet (excess AA) or during times of starvation
Some AA can be broken down into pyruvate
Many AA are broken down into Krebs cycle intermediates
How does this help?
Oxaloacetate is the second intermediate in gluconeogenesis (i.e. in the 2nd step)
Therefore, any Krebs cycle intermediate can be used to make glucose via oxaloacetate except Acetyl CoA
Glucogenic AA are AA that can make sugars
99
Molecules that can be used to make glucose
| Glycerol
(the backbone of a triacylglycerol) can be converted into DHAP and thus be used to make glucose
Gluconeogenesis is not the simple reversal of glycolysis (almost, but not quite)
All steps that are reversible go in reverse except for irreversible steps
100
What happens to lactate?
| - Option 1: cori cycle
cori cycle: the liver can take it up, convert it back to pyruvate and then do gluconeogenesis to convert it to glucose. The glucose is shipped back to the muscle.
This allows for metabolic load shifting as the liver spends the ATP and muscle generates ATP
Costs 6 ATP to make glucose from lactate -> we get 2 ATP from glucose so we are losing 4 ATP in this cycle -> metabolic loadshifting: happens when muscle needs the energy much more than the liver
101
What happens to lactate?
| Option 2
the heart can take up lactate and convert it to pyruvate and then use it for energy
Pyruvate -> acetyl CoA -> Krebs cycle
102
Why not acetyl CoA (and thus fatty acids which are broken down into acetyl CoA)? I.e. Why can I not use my fat store, the fatty acids, to make sugars?
Fats are broken down into acetyl coA
Acetyl CoA cannot be converted back to pyruvate (cannot reverse the PDC)
Acetyl CoA can’t be used to have a net gain (no gain but regenerated) in oxaloacetate in Krebs cycle
Thus, fats (with the exception of glycerol) can’t make sugars
BUT sugars can make fats (in humans) to store
103
3 key irreversible steps
All other steps are shared by both pathways
(Gluconeogenesis/glucolysis)
1. Generation of phosphoenolpyruvate (PEP)
2. Generation of fructose-6-phosphate
3. Generation of free glucose
104
Generation of free glucose
Glucose-6-phosphatase hydrolyzes the phosphate off of glucose 6 phosphate, generating free glucose and Pi
105
Generation of fructose-6-phosphate
| DRAW OUT RXN
Use fructo-1,6-bisphosphate to hydrolyze the phosphate off
No generation of ATP -> low phosphoryl transfer potential
Key regulatory step
fructo-1,6-bisPhosphatases remove phosphates from molecules by the addition of water
Then convert to glucose-6-phosphate by phosphoglucose isomerase
Could step here as the glucose remains trapped in the cell until the liver wants to secrete it
106
Generation of phosphoenolpyruvate (PEP)
In gluconeogenesis, the generation of PEP occurs in two separate steps
First: Conversion of pyruvate to oxaloacetate by carboxylation
Oxaloacetate simile has an extra carboxyl group of pyruvate from CO2 from blood
Pyruvate carboxylase adds CO2 to pyruvate, forming oxaloacetate.
In the process, 2 ATP is hydrolyzed to ADP & Pi (coupled because positive delta G)
Costs ATP
Requires a small molecules called biotin to bind and trap the CO2
ATP hydrolysis is driving carboxylation
This reaction occurs in the mitochondrial matrix, but the rest of gluconeogenesis occurs in the cytosol
Krebs cycle in matrix
107
How do you get the oxaloacetate to the cytosol since there is no such thing as oxaloacetate translocase?
Convert it to malate, transport it to the cytosol (via malate translocase) and then convert it back to oxaloacetate
This is the reverse of the first half of the aspartate/malate shuttle
108
In a totally separate view, the Generation of phosphoenolpyruvate (PEP) can be used to add carbon to a depleted Krebs cycle
CO2 was added on so that it pushes the next reaction forwad through decarboxylation which has a negative delta G -> second step of gluconeogenesis requires 2 ATP equivalence in the form of 2 GTP
Oxaloacetate is converted into phosphoenolpyruvate
Done by phosphoenolpyruvate carboxykinase
Consists of a decarboxylation driving phosphorylation
Note: this costs GTP and produces CO2
Now can continue the reverse of glycolysis until
109
Net reaction for gluconeogenesis
2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2H+ + 6H2O -> glucose + 4ADP + 2GDP + 6 P + 2NAD+
△G°’ = -38 kJ/mol
4 ATP (2 at the pyruvate carboxylase step and 2 at the phosphoglycerate kinase step)
Gluconeogenesis is expensive -> to make glucose from pyruvate costs the equivalent of 6 ATP (4 ATP and 2 GTP)
110
Regulation of gluconeogenesis
Glycolysis and gluconeogenesis is reciprocally regulated
Do not want both occurring at the same time
Regulation is at the 3 bypass reactions (these are the reactions that are unique to gluconeogenesis)
111
Regulation is at the 3 bypass reactions (these are the reactions that are unique to gluconeogenesis)
1. Fructose-1,6-bisphosphatase
2. Phosphoenolpyruvate carboxykinase
3. Pyruvate carboxylase
112
Regulation is at the 3 bypass reactions (these are the reactions that are unique to gluconeogenesis)
Fructose-1,6-bisphosphatase
Inhibited by AMP & fructose-2,6-bisphosphate
When AMP is increasing, ATP is decreasing
Activated by citrate
Citrate is the product after loading acetyl coA (then bound to oxaloacetate) into the krebs cycle
113
Regulation is at the 3 bypass reactions (these are the reactions that are unique to gluconeogenesis)
Phosphoenolpyruvate carboxykinase
Inhibited by ADP (if you do not have energy, why are you doing gluconeogenesis)
114
Regulation is at the 3 bypass reactions (these are the reactions that are unique to gluconeogenesis)
Pyruvate carboxylase
Inhibited by ADP
Just because you have lots of ATP, does not mean we have to make more glucose especially if glucose levels are high
Activated by acetyl CoA
If there is a broken krebs cycle (not converting acetyl coA into citrate) and acetyl coA builds up -> then pyruvate carboxylase is activated to convert acetyl coA to carboxylic acetate to convert oxaloacetate to put more oxaloacetate into the krebs cycle
115
Blood glucose
Maintaining blood glucose levels is critical because the brain always needs some glucose in order to survive.
The brain’s only viable fuels are glucose and ketone bodies
Fatty acids don’t cross the blood/brain barrier in high enough amounts to be a viable fuel source
116
Ketone bodies
a ketone body is the way the liver ships acetyl units (derived from acetyl CoA)
In times of starvation, the brain can retool its metabolism to use ketone bodies as an energy source
BUT, ketone bodies can only meet up to 70% of the brains energy needs; the other 30% must be from glucose
117
Blood glucose should be around _mM
5
118
High blood [glucose] (hyperglycemia)
(type II diabetes) long term over decades can lead to neurological, cardiovascular, renal and vision damage
At really high blood glucose levels (>30mM; undiagnosed type I diabetic)
Glucose can act as an osmole (molecule that controls the flow of water: it starts to pull water out of the cells
Can acutely render you unconscious
119
Low blood [glucose] (hypoglycemia)
Low blood [glucose] (hypoglycemia) can lead to an individual becoming confused, unconsciousness, and in very rare cases brain damage or death
E.g. diabetic who accident injected to much insulin
120
Red blood cells and cell of your ___ ____ also need glucose because they lack mitochondria; ...
eye lens; They go through glycolysis/fermentation and so they need glucose
121
Hormones affecting blood glucose
Insulin
glucagon
epinephrine
122
Insulin
peptide hormone released when glucose levels are high
Promotes the synthesis of glycogen, fats, and proteins
Decreases gluconeogenesis (no need to make glucose when glucose levels are high
Promotes the uptake of glucose into the cells
Most cells can’t take up glucose without insulin
Produced by Beta-islet cells of the pancreas and targets most cells in the body
123
Glucagon
protein hormone released when glucose is scarce
Must inject because it its a protein
It promotes lipolysis (release of fatty acids), gluconeogenesis, glycogen breakdown (in the liver) and protein catabolism
Promotes the release of glucose
Produced in alpha-islet of the pancreas
Primarily targets the liver adipose tissue
124
Epinephrine
A catecholamine (derivative of tyrosine) hormone released when glucose is needed in a stress situation -> Will target skeletal muscle
