Metabolism Flashcards
1
Q
What is glycolysis?
A
Glycolysis is the pathway for extracting energy from the breakdown of glucose.
2
Q
Why is glucose such a central molecule in metabolism?
A
The free energy of the breakdown of glucose into CO2 + H2O is huge! ~2840 kJ/mol (although we can only extract a small part of this energy as much is converted to heat). Besides this glucose has many possible fates in a cell:
- Its a precursor to many biomolecules like nucleic acids, polysaccharides and ECM components.
- It can be stored as starch/glycogen/sucrose
- It can be oxidized to drive the synthesis of ATP
3
Q
What is the net production of glycolysis?
A
Glucose → 2 pyruvate + 2 ATP + 2 NADH
So we get pyruvate that can be converted into acetyl CoA and go into TCA, ATP that can be used as energy and NADH that give reducing power in oxidative phosphorylation. We also get a bunch of intermediates that can be used as precursors in biosynthesis.
4
Q
What happens to the pyruvate generated by glycolysis in aerobic vs anaerobic environments?
A
In aerobic conditions, the pyruvate is converted to Acetyl-CoA by the PDH complex and feeds into the citric acid cycle. It can also go into the pentose phosphate pathway.
In anaerobic conditions, the pyruvate undergoes fermentation into ethanol in yeast and lactate in some cells (eg muscle and erythrocytes) and some microorganisms. The fermentation process oxidizes NADH, which reforms NAD+ which is cycled back to glycolysis.
5
Q
Glycolysis can be divided in two phases, which? What is different about them?
A
Glycolysis can be divided into the preparatory phase and the “pay off” phase.
- In the preparatory phase, 2 ATPs are invested (glucose → G6P and F6P → F1,6-BP) to activate the glucose and produce important intermediates. In the end of the investment phase we have 2 phosphorylated 3-C molecules instead of the 6-C start molecule.
- In the pay-off phase, 4 ATPs, 2 NADH and 2 pyruvates are generated, which make the reaction net positive.
6
Q
What is the full reaction of glycolysis?
A
glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 pyruvate + 2 NADH + 2H+ + 2 H2O + 2 ATP
with a delta free energy of -85 kJ/M - very small compared to the free energy of glucose, but most of the energy is not extracted during glycolysis, but in the ETC later.
7
Q
Go through the 10 steps of glycolysis and describe what kind of reaction each step is.
A
Investment phase:
1. Phosphorylation of carbon 6; glucose is first phosphorylated at the hydroxyl group on C-6.
- Isomerization of G6P to F6P (reversible); The glucose 6-phosphate formed is converted to fructose 6-phosphate
- Phosphorylation of F6P to F1,6-BP; this time at C-1, to yield fructose 1,6-bisphosphate. For both phosphorylations, ATP is the phosphoryl group donor.
- Fructose 1,6-bisphosphate is split to yield two different three carbon molecules, dihydroxyacetone phosphate (DHAP) and
glyceraldehyde 3-phosphate through the formation of a schiff base. This is the “lysis” step that gives the pathway its name - Isomerization of DHAP; The dihydroxyacetone phosphate is
isomerized to form a second molecule of glyceraldehyde 3-phosphate
Payoff phase:
6. Oxidation of G3P to 1,3-BPG (generating NADH); Each molecule of glyceraldehyde 3-phosphate is oxidized and phosphorylated by inorganic phosphate (via substrate level phosphorylation, high energy molecule P donor, not ATP) to form 1,3-bisphosphoglycerate.
- Phosphorylation of ADP to ATP with the phosphoryl being donated from 1,3-BPG (substrate level phosphorylation) which forms 3-PG. steps 6 & 7 are coupled and together favorable. Net, step / generates ATP and 3-PG.
- “mutase” reaction that is preparatory for next step 3-PG to 2-PG (switching places of the phosphate and OH group using His residues in active site).
- 2-PG to phosphoenol pyruvate (PEP) + H2O, which “locks” the PEP in a high energy state, if H2O didn’t leave, the molecule would immediately rearrange into a ketone.
- tautomerization of PEP (enol form) + ADP → pyruvate (keto form) + ATP practically irreversible, strongly exergonic.
Note that step 1, 3 and 10 are “irreversible” which gets very problematic when we try to go the other way around in gluconeogenesis.
8
Q
During glycolysis, all of the nine glycolytic intermediates between glucose
and pyruvate is phosphorylated. What is the three functions of the phosphoryl group?
A
The three functions of the phosphoryl group are:
1. Phosphorylated sugars can’t leave the cell (no transporters) so no further energy is necessary to retain phosphorylated intermediates in the cell, despite the large
difference in their intracellular and extracellular concentrations.
- Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. The bonds are high energy.
- Binding energy resulting from the binding of phosphate groups to the active sites of enzymes lowers the activation energy and increases the specificity of the enzymatic
reactions.
9
Q
What is gluconeogenesis?
A
The pathway for synthesizing glucose from
noncarbohydrate precursors, like pyruvate, related three- and four-carbon compounds as well as certain amino acids to glucose.
10
Q
Name three sources that can be converted into the starting material for gluconeogenesis.
A
- Lactate (animals)
- fatty acids (both)
- glucogenic amino acids like alanine. (both)
- 3PG from calvin cycle (plants)
11
Q
Where does gluconeogenesis mainly occur in mammals?
A
In the liver! It also occur to some extent in the renal cortex and in the epithelial cells that line the small intestine.
12
Q
Gluconeogenesis is not simply the reverse steps of glycolysis, why not? How is this solved?
A
Because three of the 10 reactions in glycolysis have a large negative energy change, which means that the reverse is not achievable.
This is solved by having three alternative enzymes catalyzing bypass reactions, reactions that are sufficiently exergonic to be effectively irreversible in the direction of glucose synthesis. Thus, both glycolysis and gluconeogenesis are irreversible processes in cells. In animals, both pathways occur largely in the cytosol, necessitating their reciprocal and coordinated regulation.
13
Q
Explain the first bypass reaction in gluconeogenesis.
A
As glycolysis ends with the conversion of PEP to pyruvate, we need to convert pyruvate into PEP.
Pyruvate is transported into mitochondria or is generated from alanine within mitochondria by transamination, in which the α-amino group is transferred from alanine (leaving pyruvate) to an α-keto carboxylic acid (this is also a transport mechanism for pyruvate in the blood).
a) In the mitochondria, pyruvate carboxylase adds on a carboxyl group to the pyruvate (with biotin as coenzyme) to form oxaloacetate (OAA). The role of the biotin is to covalently bind the carboxylate ion HCO3- (created in a ATP driven reaction).
b) OAA is then transported out by interconvesion to malate and in the cytosol OAA is converted to PEP catalyzed by phosphoenolpyruvate carboxykinase by decarboxylation of the added CO2- group (from step a) that rearranges electrons so that the carbonyl O attacks the gamma phosphoryl of GTP which leaves as GDP. Since CO2 leaves as a gas, this reaction leads to an increase in entropy and thus is exergonic.
14
Q
Explain the second and third bypass reactions in gluconeogenesis.
A
The second and third bypasses are simple
dephosphorylations by phosphatases (compared to the kinases that perform the corresponding steps in glycolysis)
- The F-1,6-BP to F6P reaction is catalyzed by Mg2+-dependent fructose 1,6-bisphosphatase (FBPase-1), which promotes the essentially irreversible hydrolysis of the C-1 phosphate (not phosphoryl group transfer to ADP)
Fructose 1,6-phosphate + H2O → fructose 6-phosphate + Pi - the G6P to glucose reaction is catalyzed by glucose 6-phosphatase which is a simple hydrolysis of a phosphate ester.
Glucose 6-phosphate + H2O →glucose + Pi
15
Q
What is the total energetic cost of gluconeogenesis?
A
Formation of one molecule of glucose from pyruvate requires four ATP, two GTP, and two NADH; it is expensive.
16
Q
Hexokinase, the enzyme catalyzing the first step of glycolysis (requiring ATP) exist as two different versions in mammals, Hexokinase I in muscle and Hexokinase IV in the liver. How does the regulation of these differ?
A
The hexokinase I (muscle) has a very low Km, it basically work at max speed all the time due to the high energy needs of muscle and is not tightly regulated.
The liver hexokinase on the other hand, have a much higher Km so it is regulated by substrate level and is further regulated by binding to regulatory proteins and transported into the nucleus, where it can’t act. The transport is favored by high levels of either glucose or F6P, as this indicate that the energy needs are met.
17
Q
The rapid hormonal regulation of glycolysis and gluconeogenesis is mediated by fructose 2,6-bisphosphate, how?
A
F-2,6-BP is formed by phosphorylation of
fructose 6-phosphate, catalyzed by phosphofructokinase-2 (PFK2), a regulatory pathway in response to glucagon.
If we just look at the reaction of F6P -> F-1,6-BP and the bypass reaction, PFK-1 is allosterically activated by F-2,6-BP, while allosterically inhibitory for FBPase-1 (catalyzing the bypass reaction).
When the blood glucose level decreases, the hormone glucagon signals the liver to produce and release more glucose and to stop consuming it for its own needs. The pancreas release glucagon which in the liver triggers an increase in cAMP which in turn activates PKA, which phosphorylates PFK-2 (that catalyzes formation of F-2,6-BP). With less F-2,6-BP we no longer have inhibition of FBPase-1, and thus favor gluconeogenesis. (p. 1973)
18
Q
What is glycogen?
A
Glycogen is the storage molecule of glucose in animals.
19
Q
Explain how glycogen is synthesized in short.
A
- To start glycogen synthesis, the glucose 6-phosphate is converted to glucose 1-phosphate in the phosphogluco-mutase reaction:
Glucose 6-phosphate ⇌ glucose 1-phosphate - The product is then converted to UDP-glucose by the action of UDP-glucose pyrophosphorylase, in a key step of glycogen biosynthesis:
Glucose 1-phosphate + UTP → UDP-glucose + PPi
Notice that one ATP equivalent is the cost of the formation of each UDP-glucose.
- UDP-glucose is the immediate donor of glucose residues in the reaction catalyzed by glycogen synthase, which promotes the
transfer of the glucose residue from UDP-glucose to a nonreducing end of a branched glycogen molecule, forming an (α1 → 4) linkage. - A “branching enzyme” transfers 6-7 glycogen units from the non reducing end to a C-6 of another unit, creating (α1 → 6) branches.
20
Q
Explain the process of glycogen degradation.
A
Glycogen degradation is catalyzed by glycogen phosphorylase, which breaks the (α1 → 4) bonds with Pi as nucleophile to reform G1P, which shortens the glycogen chain by one residue. The G1P is then either phosphorylated into G6P and used in glycolysis or into glucose and exported where it’s needed.
A debranching enzyme transfers branches to the non-reducing end which is in turn broken down by the glycogen phosphorylase reaction described above.
21
Q
Pyruvate is a central metabolite that links glycolysis and the citric acid cycle. Explain the reaction catalyzed by the PDH complex in short.
A
The overall reaction of the PDHc is a oxidative decarboxylation, pyruvate is first decarboxylated, and then linked to CoA in a transacetylation reaction through a thioester linkage, where the acetyl group is considered “activated” as the hydrolysis of thioesters have a large negative free energy.
The reduced enzyme components are reoxidized by NAD+ which gets reduced to NADH with reducing power that can be utilized elsewhere in the cell.
22
Q
What is the overall production of the citric acid cycle?
A
In the TCA, acetyl-CoA undergoes a series of redox reactions that harvests the bond energy in the form of NADH, FADH2 and ATP molecules.
Overall, one turn of the citric acid cycle releases two carbon dioxide molecules and produces three NADH, one FADH2 and one ATP or GTP. The citric acid cycle goes around twice for each molecule of glucose that enters cellular respiration because there are two pyruvates—and thus, two acetyl-CoAs made per glucose. So the net production per glucose is 6 NADH, 2FADH, 2ATP/GTP.
Note: since the reaction catalyzed by PDHc also produces 1 NADH per pyruvate, you could consider those too.
23
Q
Three of the eight steps of the citric acid cycle are tightly regulated, why these? What kind of regulation is the most common?
A
The three tightly regulated steps (1,3 and 4) are the irreversible ones. Irreversible reactions are generally the ones being regulated, as energy is wasted if irreversible reactions that are not needed take place. Reversible reactions don’t require an energy investment.
The regulation is mostly allosteric, where molecules indicating a low energy state activates (ADP, Ca2+) and products and molecules indicating a well fed state (ATP) inhibits. For the first step (Ac-CoA to citrate), high concentrations of later products in the cycle inhibits as well, such as succinyl-CoA, as this indicates that the cycle is running fine.
24
Q
The first reaction of the citric acid cycle is the reaction it’s named after. Explain the reaction mechanism in short.
A
The first step, catalyzed by citrate synthase, is a Claisen condensation. In the active site, the two catalytically active residues are a deprotonated Asp and a His.
- When Acetyl-CoA binds, the Asp takes up a hydrogen from the methyl group and with electron delocalization, the carbonyl oxygen oxidizes the His residue and forms an enol intermediate stabilized by hydrogen bonding with the His.
- The enol intermediate rearranges to attack the carbonyl carbon of OAA, with His positioned to retake the proton it previously donated. The carbonyl oxygen on OAA is protonated by another His residue and the condensation generated citroyl-CoA.
- The thioester linkage is then hydrolyzed so that CoA leaves and citrate is formed.
(Step two is a isomerization catalyzed by aconitase (FeS cluster as cofactor) to form Isocitrate (switching places of the H and OH group) to facilitate the first decarboxylation (step 3)).
25
What is the products and enzyme in the third step of the TCA cycle?
Step 3 is catalyzed by isocitrate dehydrogenase, where isocitrate is first oxidized by NAD(P)+ which forms NAD(P)H + H+, then decarboxylated (CO2 leaves) facilitated by Mg+ and then the enol intermediate is rearranged to generate α-Ketoglutarate.
26
The fourth step of the TCA cycle is the last irreversible step, describe it in short.
Step four is catalyzed by the α-ketoglutarate dehydrogenase complex in a reaction homologous to PDHc, a decarboxylative oxidation of α-ketoglutarate, which forms Succinyl-CoA, CO2 and NADH. (ΔG′° ≈−33.5 kJ/mol)
27
Step five of the TCA cycle is the ATP/GTP generating step. What drives the synthesis and how does the reaction work in short?
Hydrolysis of the thioester bond in Succinyl-CoA have a large negative free energy (ΔG′° ≈−35 kJ/mol) which drives the synthesis of a phosphoanhydride bond in in either GTP or ATP, with succinyl being formed in the process.
First, an inorganic phosphate is added in the place of CoA, then the Pi is taken up by a His residue in the active site, to then be transferred to GDP or ADP to form GTP or ATP.
28
Step 6-8 of the citric acid cycle are a series of reactions with a specific purpose, what? What is generated in the process?
The purpose of step 6-8 is to regenerate the OAA from succinate so that a new cycle can begin. Its a oxidation sequence (succinate - fumarate - L-malate -> oxidation to OAA) that generates an FADH2 (step 6) and one NADH (step 8).
29
Name the three steps of the TCA cycle where NADH is formed.
- Step 3, Isocitrate to α-ketoglutarate.
- Step 4, α-ketoglutarate to Succinyl-CoA
- Step 8, L-malate to OAA.
30
All in all, only two ATP (or GTP) are formed from one glucose (one from each pyruvate/acetyl-CoA) in the citric acid cycle, but the number of ATPs ultimately formed from the glycolysis-PDHc-TCA is much more, how many?
30-32 ATPs!
31
The citric acid cycle is not only a process that harvests energy from pyruvate, but also functions as a hub of metabolism, with catabolic pathways leading in and anabolic pathways leading out. Name tree key intermediates in the CA cycle, and what they can be used for.
- Citrate is a precursor for fatty acids and sterols.
- both α-ketoglutarate and Succinyl-CoA are precursors for amino acid synthesis, heme and nucleotides (purines).
- OAA is also a precursor for amino acid synthesis and nucleotides (pyrimidines)
32
Because so many of the intermediates in the citric acid cycle are used in biosynthesis, the withdrawal could potentially cause the cycle to stop running or slow down. What has the cell done to solve this?
When the withdrawal of cycle intermediates for use in biosynthesis lowers the concentrations of citric acid cycle intermediates enough to slow the cycle, the intermediates are replenished by anaplerotic reactions (Greek, “to refill”). These are for example:
- pyruvate to OAA in liver/kidney
- PEP to OAA in heart/skeletal muscle
- PEP to OAA in plants
- Pyruvate to malate in bacteria and eukaryotes.
33
The energy harvested through oxidation in glycolysis, the conversion of pyruvate to Acetyl-CoA and the citric acid cycle culminates in oxidative phosphorylation in the electron transport chain. Where is it located in eukaryotes and bacteria?
Oxidative phosphorylation happens in the inner membrane of mitochondria in eukaryotes and the inner membrane of bacteria. The outer membrane of mitochondria is porous so "small" molecules and ions can diffuse through, while the inner membrane is very tight and contains transporters for everything that should be taken in/out.
All the processes of energy catabolism (glycolysis, PDHc, the citric acid cycle and oxidative phosphorylation) take place in the mitochondria besides glycolysis, which takes place in the cytosol.
34
Oxidative phosphorylation begins with the entry of electrons into the series of electron carriers called the respiratory chain. Most of these electrons arise from the action of dehydrogenases that collect electrons from catabolic pathways and funnel them into universal electron acceptors — nicotinamide
nucleotides (NAD+ or NADP+) or flavin nucleotides (FMN or FAD). Explain the mechanism of action for NAD+ and FAD.
NAD+-linked dehydrogenases remove two hydrogen atoms from their substrates. One of these is transferred as a hydride ion (:H−) to NAD +, and the other is released as H+ in the medium.
The oxidized FAD can accept either one electron (yielding the semiquinone/radical form) or two (yielding FADH2). Electron transfer occurs because the flavoprotein the FAD is attached to has a higher reduction potential than the compound oxidized.
35
In addition to FAD and NAD+, three other electron carriers are present in the respiratory chain, which? Describe them briefly.
- Ubiquinone (also called coenzyme Q,
or simply Q) can accept one electron to become the semiquinone radical (∙QH) or two electrons to form ubiquinol (QH2). It's a small lipid soluble molecule that can move freely between the two lipid layers that constitute the inner membrane.
- Cytochromes: Proteins with bound heme groups (a & b bound tightly but not covalently, c bound covalently). The iron in the heme groups are redox active in their oxidative state Fe2+ or Fe3+.
- Iron-sulfur proteins: proteins containing FeS clusters that almost exclusively exist to transfer electrons. Many different compositions of Fe and S.
36
The electron transfers happen in protein complexes embedded in the inner membrane, name them.
Complex I: NADH dehydrogenase (Fe-S)
Complex II: Succinate dehydrogenase (Fe-S)
Complex III: U:C oxidoreductase + Cyt. c (Heme and Fe-S)
Complex IV: Cytochrome oxidase (Heme and Cu)
All have different structure and size, but similar function, transferring electrons trough electron carriers to finally reduce O2.
37
Describe the reaction mechanism in complex I of the ETC.
Complex I catalyzes the transfer of a hydride ion (:H−) from NADH to FMN (analog to FAD). From the FMN, two electrons pass through a series of Fe-S centers to ubiquinone, which forms QH2, which diffuses into the lipid bilayer.
The complex is a proton pump which transfers 4H+ from the N side (matrix), to the P side (intermembrane space). This reaction is very energy demanding as the proton flux produces an electrochemical potential across the inner mitochondrial membrane. The free energy from the redox reaction facilitates this.
In short: 2 electrons donated from NADH as a hydride ion to Ubiquinone, the redox driving 4H+ to be pumped out into the intermembrane space.
38
Describe the reaction mechanism in complex II in the ETC.
Complex II catalyzes the oxidation of Succinate to fumarate, and the electrons is taken up by FAD which is reduced to FADH2, the two electrons is then transferred from FADH2 to Q through Fe-S clusters to finally reduce ubiquinone (Q) to QH2. This complex doesn't directly contribute to the electrochemical potential across the membrane (not a H+ pump)
39
Describe the reaction mechanism in complex III in the ETC.
Complex III catalyzes the transfer of electrons from QH2 to cytochrome C, one at a time, each resulting in 2 H+ being transferred to the P side. At this stage we have moved 8 H+ over to the P side.
To ensure that the semiquinone radical (∙QH) doesn't cause damage, "Q cycling" is utilized.
40
Describe the reaction mechanism in complex IV in the ETC.
Complex IV is also a proton pump (2H+ net (two H+ added to matrix by 2 NADH)). The 4 electrons from Cyt. C comes into the Cu-A center, goes through a heme center and moves to the Cu-B center in which the electrons are used to reduce 1/2 O2 to 1 H20 (two electrons for each oxygen)
Some problematic intermediates are generated in this reaction, ∙OH- and H2O2, which can form ∙OH (ROS) that mitochondria needs to deal with.
41
What is the summarized reaction of the electron transport chain?
NADH + 11 H+(N) + 1/2 O2 --> NAD+ + H2O + 10 H+(P)
So we have moved 10 protons from the N side to the P side, creating a charge difference of 20 units. The H+ transfer creates a gradient, both concentration (ΔpH) and charge (inside negative) and according to the chemiosmotic model, this creates a proton motive force (PMF) which drives APT synthesis.
42
What is the ATP equivalent of the 10 H+ transferred to the P side in the ETC?
10 H+ transferred to the P side = around 6.5 ATPs!
43
Which of the four complexes in the ETC act as proton pumps?
I, III and IV.
44
Explain how ATP synthesis work.
ATP synthesis depends on and is coupled to the H+ transfer across the inner mitochondrial membrane (the whole ETC). As H+ (P) are funneled back to the N side, it drives the catalysis of ADP to ATP. Phosphate is transported into the matrix through a symport with H+, called phosphate translocase. ADP is transported into the matrix and ATP is transported out by an antiport.
45
There are no transporters from OAA in the inner mitochondrial membrane, how is it transported?
Through the malate aspartate shuttle.
46
Why does electron transfer stop in the absence of ATP synthesis?
The gradient becomes to large to overcome?
47
In total, how much of the total free energy of glucose is taken up by metabolism?
About 34%, which seems low, but we need heat (which makes up the rest) so not bad overall!
48
Where does the photosynthetic reactions occur in eukaryotes vs bacteria?
Photosystems are found in the thylakoid membranes of plants, algae, and cyanobacteria. These membranes are located inside the chloroplasts of plants and algae. Photosynthetic membranes in prokaryotes, by contrast, are not organized into distinct membrane-enclosed organelles; rather, they are infolded regions of the plasma membrane. In cyanobacteria, for example, these infolded regions are also referred to as thylakoids.
49
Photosynthesis consists of two reactions, which?
- The light dependent reactions: which generate ATP and NADPH ( +O2), from which the NADPH is used in the:
- Carbon fixating reactions (CTF) which uses ATP to fixate CO2 into carbohydrates.
50
Explain the structure of chloroplasts and name the most important components.
Chloroplasts have a permeable outer membrane and a tight inner membrane folded into thylakoids. The thylakoids are packed into stacks called grana. The inside of the thylakoid membrane is called lumen (P side) and the intermembrane space is called the stroma (N side).
51
Explain in short how the light (photon energy) is absorbed.
The chloroplasts contain light harvesting complexes comprised of highly organized pigment molecules, like chlorophyll, beta-carotene and lutein (xanthophyll). When photons hit these pigment molecules, they excite electrons that excite a new one in a surrounding molecule and so on until it reaches a reaction center. The reaction center contains a specialized pair of chlorophyll, which get enough energy for an electron to be transferred to an acceptor molecule that is part of the photosynthetic electron-transfer chain. The energy
is used to create a charge separation that initiates electron flow through a series of oxidation-reduction reactions that drive the reduction of NADP+ to NADPH.
The chlorophyll in the reaction center becomes a radical but receives an electron from a nearby donor which become positively charged.
52
Bacteria have two types of reaction centers, which is present in what type of bacteria?
Type II reaction centers are present in purple bacteri, which have a bc1 cytochrome complex similar to complex III in the eukaryotic electron transport chain. The reduction of the bc1 complex moves 4H+ to the P side, which drives ATP synthase.
Type I reaction centers are present in green sulfur bacteria, and is similar to type II but distinct. The reduction of the bc1 complex moves 4H+ to the P side, which drives ATP synthase AND electron transfer to ferredoxin drives the reduction of NAD+ to NADH (note that this is unique to sulfur bacteria as cyanobacteria and plants reduce NADPH).
53
Plants and cyanobacteria have the same setup for the electron transfer. Explain it in short.
The light harvesting complexes transfers the photon energy to photosystem II, which excites an electron in cyt P680 to be transferred to pheophytin --> plastoquinone A --> plastoquinone B --> cyt b6f complex which pumps H+ into the lumen.
The electron missing in cyt P680 is replenished by H20 as donor to form O2.
From the cyt b6f complex the electron is transferred to photosystem I via plastocyanin which is in turn excited by photon energy to move down an electron transport chain ultimately through ferredoxin that lead to the reduction of NADP+ to NADH.
54
Photosystem I and II are physically separated in the thylakoid membrane, how and why?
PSII (bigger) and LCHII are located in the grana stacks, while PSI (smaller) is located in the non-appressed membranes. The reason for the separation is to prevent "exciton theft" (exciton=excited electron). If they were connected, PSI would steal the light as it has lower reduction potential and therefor requires lower energy photons to be excited than PSII.
55
But the problem of "exciton theft" should disappear in strong sunlight right? as the light should be enough for both.
Yes! And they have solved this one by phosphorylation/dephosphorylation of the LHCII which links together the grana stacks. Phosphorylation of Thr residues on LCHII resolves the grana stacks and unlinks them, so that everything mix. Dephosphorylation reverses this.
56
What prevents back flow of H+ in plants?
Retinal! Retinal has one structure in darkness and another in light, so during the day, when the light dependent reactions are running, retinal isomerizes to a structure with a lower pKa, which lowers the proton transfer rate and thereby limits back flow.
57
Compare mitochondria, thylakoid and bacteria in terms of the proton flow direction.
In mitochondria and in bacteria, the protons are pumped outwards (into the intermembrane space) but in thylakoids, the protons move in the other direction, from the stroma into the lumen (the innermost compartment).
58
What types of strategies can be used to drive substrate level phosphorylation (not ATP as donor)?
One example of a substrate level phosphorylation reaction is step 7 in glycolysis, 1,3-BPG donating one of its phosphoryl groups to ADP, forming ATP and 3-PG. The strategy for driving this phosphorylation is that the 1,3-BPG is an "activated ester" meaning that the phosphate attached to the carboxyl carbon is more reactive as it is attached to a good/stable leaving group: carboxylate. Because of this, the phosphoryl can act as a nucleophile and attack ADP, even though it is generally a very bad nucleophile.
Another example of a strategy is using the stability of the product to drive substrate level phosphorylation, as in the last reaction of glycolysis: PEP + ADP → pyruvate + ATP in which the product formed is first pyruvate in enol form, which then spontaneously and non-enzymatically converts to its keto form (tautomerization). The tautomerization provides enough free energy to drive the substrate level phosphorylation.
59
Discuss some advantages and disadvantages of the compartmentalization (mainly in eukaryotes) of the different processes of glucose breakdown to ATP synthesis.
Advantages:
- With compartmentalization you get more opportunity for regulation.
- Protection from side reactions
- Under changing conditions (like anaerobic/aerobic) the separation make it easy to "switch" routes.
Disadvantages:
- Need for transport of metabolites cost energy
Possible causes; could just be an evolutionary remnant, also the independent replication of mitochondria give the possibility to have different amounts of them in different cell types.
60
The electron transport chain is always in a tight membrane, why?
Because you need a barrier to create the charge separation driving ATP synthesis, and the lipid soluble carriers need a non-polar environment to move around in, so lipid bilayers are perfect!
61
What would be a plausible explanation to why the Quinone structure is so well conserved across all different organisms?
Quinone is an electron carrier in the ETC, that carries electrons donated from NADH/FADH2 but the acceptor atom (Fe in Cytochrome and Cu in plastocyanine) only takes up one electron at a time, which forms a radical. Quinone is structured so that the radical can move around on different atoms, which makes it very stable even though its in a radical form.
62
What sources of lipids/fatty acids are there?
Lipids/fatty acids can either be ingested as food or can come from stored fat in adipocytes.
63
What happens when fatty acids/lipids are ingested? How are they taken up in the body?
1. since fatty acids are poorly soluble in water, we need something to make them more soluble. Bile salts/acids are squirted out in the intestine from the gallbladder, which emulsify the fatty acids (make them more soluble.
2. Intestinal lipases breaks down the triacyl-glycerols info free fatty acids that can be transported into intestinal cells via mucosa.
3. The free fatty acids and other breakdown products are taken upp and re-esterified to triacylglycerols.
4. The triacylglycerols are bound in water soluble complexes with cholesterol and apolipoproteins called chylomicrons.
5. The chylomicrons move through the bloodstream and lymphatic system to be transported to different tissues.
6. Another lipase breaks down the chylomicrons and its triacylglycerols to free fatty acids again and is taken up by tissues.
7. In the tissues, the fatty acids are either oxidized as fuel or re-esterified for storage. Note, all cells can't use fatty acids for fuel, eg the brain cannot, but muscle/liver cells can for example.
In this process, the fatty acids are converted between their water soluble and non-soluble form for uptake and transport purposes.
64
What part of the triacyl glycerols can be used for glucose synthesis?
The fatty acid chains can not be used for glucose synthesis, but the glycerol skeleton can! By adding a phosphate group (kinase) and then oxidize the central carbon (removing two hydrogens) (dehydrogenase and NAD+) we get a ketone (now we are already in the gluconeogenesis) and by an isomerase we get an aldehyde. Then we can merge one of the 3-C ketones and one 3-C aldehyde to get fructose-1,6-BP which can move upwards in gluconeogenesis.
So glycerol is a decent fuel, but because it's such a small part of a fat molecule it's not nearly as potent as a fuel as glucose.
65
Where does fatty acid breakdown occur in eukaryotes?
beta-oxidation occurs in the mitochondrion in yeast/animals (peroxisome in plants). The end product of beta oxidation is acetyl-CoA, which can go right into the citric acid cycle, which also happens in the mitochondria, so it is very convenient that everything happens in the correct compartment from the beginning.
66
How does the fatty acids get into the mitochondria?
Short- and medium-chain fatty acids, those with chain lengths of 12 or fewer carbons, enter mitochondria without the help of membrane transporters.
Long-chain fatty acids, those with 14 or more carbons, which constitute the majority of the FFAs obtained in the diet or released from adipose tissue, cannot pass directly through the mitochondrial membranes: they must be transported through the carnitine shuttle.
67
Explain the reaction of activating long fatty acids to enable transport into mitochondria.
In order for long chain fatty acids to be transported into mitochondria, they need to be activated to fatty acyl–CoA in a reaction catalyzed by fatty acyl-CoA synthetase.
1. The carboxylate ion in the end of the fatty acid chain attacks a phosphate in ATP and form a fatty acyl-adenylate (mixed anhydride) and pyrophosphate (PPi). The PPi is immediately hydrolyzed to two Pi, which releases energy that is used to drive the next step.
2. The thiol group (SH) of Coenzyme A attacks the acyl-adenylate, breaking the C-O bond forming AMP and Fatty acyl CoA (thioester).
The now activated fatty acid can now be transported into the mitochondria through the carnitine shuttle.
68
Explain the reactions of the carnitine shuttle in short.
Because there are no transporters for fatty acyl-CoA, another re-esterification step to a carrier is needed.
CoA is exchanged for Carnitine (essentially the structure of an amino acid) by carnitine acyl-transferase I and the carnitine ester is transported through the membranes via transporters, and the carnitine is then exchanged for a CoA inside the mitochondria.
Note, the carnitine acyl-transferase is involved in the regulation of beta-oxidation.
69
Explain the overview of how beta oxidation is used to fuel the cell.
In beta oxidation, two carbon fragments are "chopped off" the chain one at a time, starting in the carboxyl end to yield acetyl-CoA that go into the citric acid cycle. The electrons from both the beta oxidation and the citric acid cycle are carried on NADH and FADH2 to the ETC to drive ATP synthesis.
70
Describe the steps of beta oxidation of a 16-C fatty acid (saturated), like palmitoyl.
For a 16 carbon fatty acid, we need seven beta oxidations, which yields 8 Acetyl-CoA.
1. Both the alfa and beta carbons are oxidized, which forms a double bond between them (trans) and the electrons are donated to FAD to form FADH2. (reaction very similar to the reaction catalyzed by succinate dehydrogenase, step 6, in the CA cycle)
2. The next step is to add in a hydroxyl (OH) group to the beta carbon, catalyzed by a hydratase. In the reaction, water is added, to form a hydroxyl group on the beta carbon and one hydrogen is added to each of the double bound carbons, breaking the double bond (similar to step 7 of the CA cycle).
3. The beta carbon is then oxidized, and the electrons are transferred to NAD+ to form NADH + H+, resulting in a ketone on the beta carbon, a beta-ketoacyl-CoA (again, reaction is analogous to step 8 of the Ca cycle, where malate is oxidized to form OAA).
The C-C bond between the methylene and the two ketones are fairly weak, and its hydrogens are quite acidic as the negative charge remaining after deprotonation can delocalize to either of the ketones. The weak nature of these bonds is crucial for the subsequent cleavage.
4. The thiol group of CoA attacks the beta carbon, breaks the bond between the methylene and the beta carbon, releasing Acetyl-CoA, catalyzed by thiolase. The 16 carbon chain is now 14 carbons long ((C14)-acyl-CoA) and the next beta oxidation can now occur.
A 16 carbon chain being oxidized and the resulting Acetyl-CoAs being oxidized in the CA cycle leads to 108 ATPs being ultimately formed! A LOT.
Remember! The example here is a fully saturated fatty acid, but many fatty acids are unsaturated, so there are some tweaks to this process for unsaturated fatty acids.
71
The reaction sequence of beta oxidation is analogous to the three final steps in the citric acid cycle, but also to the oxitation of some amino acids. Review these steps in short.
In all three of these reactions, the starting point is a molecule with two methylene (CH2) groups with a single bond in between.
1. Reduction leads to a double bond forming between the two carbons.
2. Hydration (addition of water) yields an hydroxyl group on one carbon and the other becomes a methylene again when the double bond is broken.
3. Oxidation yields a ketone where the hydroxyl previously was.
(in beta ox: 4. CoA attacks the beta carbon)
This process releases electrons to be used in the ETC to drive ATP synthesis.
72
What is formed in one pass of beta oxidation?
one Acetyl-coA, one fatty-acyl-CoA that is two carbons shorter, two pairs of electrons (one pair on NADH and one pair on FADH2) and 2 H+.
73
Since the trans bond between the alpha and beta carbon is a requirement for enoyl-CoA hydratase to add the H2O, how is this solved for unsaturated fatty acids?
Two additional enzymes are present in the mitochondrial matrix to resolve this problem. First you run beta oxidation as usual until you come to the cis bond. The first is an isomerase that isomerizes the molecule to a trans version, that can then be acted on by the enoyl-CoA hydratase as usual. This in enough for monounsaturated fatty acids, but for polyunsaturated ones we need an additional enzyme, a reductase.
Note: Normally you need to reduce the alpha and beta carbon to get to the trans bond formation, but with this, you don't need to, so you miss out on one FADH2 being formed. Therefore you get less energy out of unsaturated fatty acids.
If we have two sets of unsaturated carbons with one saturated in between for example, first, the isomerase isomerizes one of the double bonds to the trans form, while the reductase reduces two of the methylene's (with electrons donated from NADH), so that only one double bond is left. The isomerase can then move the double bond (still trans) so that it sits in between the alpha and beta carbon.
Note here that we actually "spend" electrons by using one NADH to donate electrons, so we get even less energy out.
74
Some plants produce fatty acids with an uneven number of carbons, how are these handled in beta oxidation?
For fatty acids with an uneven number we'll get to the point when there are three carbons left, and since the methyl group is too stable, we can't just chop it off to form Acetyl-CoA. Instead, we add on a carbon from biotin catalyzed by a carboxylase, move the CoA to form succinyl-CoA which enters into a different part of the citric acid cycle, step 5, where it can be oxidized.
75
What are ketone bodies?
Ketone bodies are an alternative (emergency) fuel molecule (much less favorable form of energy) and a way to transport Acetyl-CoA to tissues with high energy needs, like the brain.
76
What are the two ketone bodies called and how are they synthesized?
The two ketone bodies are called acetoacetate and β-hydroxybutyrate.
Ketone bodies are synthesized from two Acetyl-CoA molecules.
1. Thiolase catalyzes the formation of a β-Ketoacyl-CoA (The same enzyme that catalyzes the last step of β-oxidation, but the reverse reaction) removing one CoA.
2. Another acetyl group is added, from Acetyl-CoA (2 more carbons added) and the other acetyl-CoA from the beginning is hydrolyzed, to form acetoacetate (the first ketone body).
Acetoacetate however, contains a methylene group in between two ketones, which means the bonds are pretty easily broken. If the bond breaks, it becomes decarboxylated and form acetone which has no metabolic value. To minimize this happening, an extra step occurs:
3. Acetoacetate can be reduced by a dehydrogenase, to form β-hydroxybutyrate, which no longer have the methylene in between ketones, so it's a lot more stable (and more water soluble) and can be transported through the blood to the brain for example, where it can be converted into acetyl-CoA as fuel.
77
When the ketone bodies reach the target tissue, how is it converted to Acetyl-CoA?
The conversion is essentially just the backwards reaction of their formation:
1. The β-hydroxybutyrate is oxidized to form Acetoacetate
2. Succinyl-CoA donates its CoA to form acetoacetyl-CoA.
3. Thiolase catalyzes the attach of CoA to the ketone carbon and two Acetyl-CoA are formed.
78
How is fatty acid oxidation regulated by blood glucose levels?
Fatty acyl–CoA formed in the cytosol has
two major pathways open to it: (1) β-oxidation by enzymes in mitochondria or (2) conversion into triacylglycerols and phospholipids by enzymes in the cytosol. Once fatty acyl groups have entered the mitochondrion, they are committed to oxidation to acetyl-CoA, which consumes a precious fuel, so it is tightly regulated.
The rate limiting step for β-oxidation is the carnitine shuttle, and that is where the major regulation occurs. A precursor in fatty-acid synthesis, Malonyl-CoA, inhibits the carnitine transferase responsible for the transport.
The synthesis of malonyl-CoA is linked to glucose levels: high concentrations of glucose in the blood causes insulin to rise, which activates a phosphatase that dephosphorylates (which activates) the enzyme catalyzing malonyl-CoA synthesis, leading to upregulation of the synthesis of malonyl-CoA, which inhibits β-oxidation. Low glucose levels instead cause glukagon levels to rise, which activates a kinase that phosphorylates the malonyl-CoA synthesizing enzyme (inactivating it) leading to activation of β-oxidation.
79
What is the fate of fatty acids in hepatocytes?
Fatty acids are converted to Acetyl-CoA and from there either:
- to ketone bodies (at low blood glucose levels) or the
- Goes into the citric acid cycle and OAA is converted into glucose.
Denna är sjukt oklar, kolla kontext på denna, känns som att det borde vara vid ett visst state?
80
Explain how the synthesis of fatty acids reaction works.
Fatty acid synthesis is not the reversal of β-oxidation! Synthesis occurs in the cytosol, while β-oxidation happens in mitochondria, so the processes are separated physically (probably both due to regulation and energy conservation purposes)
The precursor for FA synthesis is malonyl CoA and fatty acid synthase catalyzes the reaction, a multienzyme complex
1. The malonyl group (COCH2COO-) is decarboxylated and an acetyl group (COCH3) is added in a condensation reaction to form a double ketone (like in the next last step in β-oxidation)
2. The acetyl derived ketone is then reduced to form a hydroxyl group on the acetyl carbon. (NADPH + H+ as donor).
3. A dehydration forms a trans double bond like that in β-oxidation.
4. Another reduction results in the carbons being saturated, and the acyl group have been lengthened by two carbons.
This is repeated so that two carbon fragments are added. An Acyl carrier protein (ACP) holds on to the growing acyl chain via a thiol ester. When the structure no longer fit in to the multienzyme complex it stops.
81
Phospholipids are one of the most important component of biological membranes, which in turn are the most important component of cells. How are phospholipids synthesized?
The building blocks for phospholipids are Acyl-CoA and L-glycerol-3-phosphate. The enzyme Acyl transferase use ATP to cleave the thioester bond and attach one acyl to each of the two free hydroxyl groups of the glycerol part of the L-glycerol-3-phosphate to form a phosphatidic acids .
Different head groups can then be attached to the phosphate group of the phosphatidic acids to form different types of glycerophospolipids, (or be converted to triacylglycerols)
82
What are Eicosanoids?
Eicosanoids are derivatives of arachidonic acid, a poly-unsaturated fatty acid containing 20 carbons and with double bonds at the 5, 8, 11, and 14th carbon, which gives it a drawn out "C" structure.
83
Name two eicosanoids.
Some examples are:
- Prostaglandins
- Prostacyclins
- Thromboxanes
- Leukotriene
- Lipoxins
84
Name three things in the body that eicosanoids are responsible for.
- Inflammation
- Fever
- Pain
- Blood pressure regulation
- regulation of sleep/awake cycle
(Involved in asthma, rheumatoid arthritis etc.)
All eicosanoids act by binding to GPCRs, which triggers the release of 2nd messengers (often cAMP)
Because of their involvement in inflammation and pain, they are common targets for drug development, eg NSAIDs (Non-steroidal anti-inflammatory drugs).
85
What enzyme catalyzes the formation of the precursor to many eicosanoids? What is the precursor called?
PGH synthase is the enzyme that catalyzes the synthesis of PGH2 from arachidonic acid. PGH2 is a precursor for ALL prostaglandins, prostacyclins and thromboxanes.
86
Why can drugs targeting eicosanoid synthesis be problematic?
PGH synthase is inhibited by NSAIDs, which means no PGH2 is formed. Even though some eicosanoids cause symptoms that are "bad" some eicosanoids are responsible for important processes, like blood clotting and pressure. Taking NSAIDs for too long can thin out blood.
87
How does aspirin work to inhibit eicosanoid synthesis?
Aspirin bind to the hydroxyl group of Ser530 of PGH synthase, blocking the active site so that it can't form PGH2.
88
Amino acid metabolism consist of many intertwined processes, but one step can be considered as the key part, which?
All of the pathways for amino acid degradation include a key step, always involving a pyridoxal phosphate cofactor, in which the α-amino group is separated from the carbon skeleton and shunted into the pathways of amino group metabolism. The carbon skeletons are broken down to citric acid cycle intermediates.
89
Amino acids undergo oxidative degradation in three different metabolic circumstances, which?
1. When amino acids released during normal protein turnover are not needed for new protein synthesis.
2. When ingested amino acids exceed the body’s needs for protein synthesis.
3. cellular proteins are used as fuel because carbohydrates are either unavailable or not properly utilized due to starvation or uncontrolled diabetes mellitus.
90
Degeneration of amino acids generate an amino group NH4, which is toxic in free form. But, as nitrogen is hard to come by it needs to be handled carefully. What are the main fates of the amino group?
Amino groups can be used for biosynthetic purposes or as energy in gluconeogenesis, but in excess, it needs to be excreted in a safe way.
91
How is nitrogen excreted in mammals, fish and birds?
Mammals: urea H2N-C(=O)-NH2.
Fish: ammonia (NH4+)
Birds: Uric acid.
92
The liver in central in amino acid metabolism, what happens to the amino acids in hepatocytes?
In the liver, the amino acids are converted into their alpha-ketoacid form (transamination), releasing the amino group and can then be converted into pyruvate to be used for gluconeogenesis. The amino group can then be excreted or used in biosynthesis.
93
How is the low pH of the stomach related to the degradation of protein?
The low pH protonate the side chains of proteins, which causes them to loose interactions with other side chains and ultimately loose their tertiary structure. You also protonate the peptide bond, which makes it a lot easier to break for the proteases.
94
Explain the process of transamination in short.
Transamination occurs in the liver, and is catalyzed by amino transferase. Amino transferase use pyridoxal phosphate (PLP) as cofactor to move the amino group from the amino acid to α-ketoglutarate to form glutamate, which converts the amino acid to an α-ketoacid.
95
Once we have the amino group on glutamate, we want to transfer it further to excrete of use, how?
To transport it, we need to convert it to glutamine, which functions as amino group transport in blood and amino group donor in biosynthesis (eg nucleotides).
The conversion is done by glutamine synthetase (requires ATP) which adds on an amino group (−NH 2). If the amino group is to be excreted, the glutamine is then converted to glutamate again in the liver and the NH4+ is converted to urea.
96
The transport of the amino groups from muscle has a special carrier, which?
Alanine! Through glycolysis in the muscle, pyruvate is created, and alanine aminotransferase then moves the amino acids from glutamate to pyruvate to form Alanine (and α-ketoglutarate) which goes through the blood to the liver, and is there transaminated to pyruvate again which can be used in gluconeogenesis and the amino group is excreted.
97
Explain the urea cycle in short.
The urea cycle consists of five steps:
1. Ammonia (NH4+) is moved from glutamate to carbamoyl phosphate (precious, eg used in pyrimidine synthesis) in the mitochondria.
2. Formation of citrulline from ornithine and carbamoyl phosphate (entry of the first amino group in the cycle); the citrulline passes into the cytosol.
3. Citrulline + asp --> formation of argininosuccinate through a citrullyl-AMP intermediate (entry of the second amino group).
4. Formation of arginine from argininosuccinate (cleavage); this reaction releases fumarate, which enters the citric acid cycle.
5. Formation of urea; Arginine hydrolase cleaves off urea from arginine, this reaction also regenerates ornithine that is transported back to the mitochondrial matrix and this completes the cycle.
98
Why is urea a good excretion molecule?
- Urea is very soluble in water, each H can make two hydrogen bonds and the O can make two, so 6 in total. It's a perfect hydrogen bond donor/acceptor.
- The symmetric structure of the molecule, with three highly electronegative atoms evenly distributed leads to an even electron distribution, so it's very stable and unreactive.
99
What are the carbon skeleton of amino acids used for in amino acid degradation?
The carbon skeletons are used to produce intermediates in the citric acid cycle, eg aspartate --> OAA or Isoleucine --> Succinyl-CoA.
100
What are nucleotides degraded to?
Purines --> uric acid
Pyrimidines --> Succinyl-CoA
101
How is ammonia incorporated into
biomolecules by bacteria? How is the process regulated?
The enzyme glutamine synthetase is central in the assimilation on NH4+ into biomolecules.
Glu + ATP + NH4+ --> Gln + ADP + Pi + H+
The process is tightly regulated, both allosterically and by covalent modification via adenylation. It is mainly positively regulated by ATP and negatively regulated by many different inhibitors, like gly, ala, trp, his, carbamoyl phosphate (from the urea cycle) and AMP (essentially all compounds containing nitrogen).
Since the reaction requires ATP and the products of glutamine are expensive to make, it makes sense that this is such a regulated process.
102
Nucleotides can be synthesized via two pathways, which? Explain what the building blocks are in each.
- The de novo pathway: begins with their metabolic precursors: amino acids, ribose 5-phosphate, CO2, and NH3.
- The "salvage" pathway: recycles the free bases and nucleosides released from nucleic acid breakdown.
Both types of pathways are important in cellular metabolism! An amino acid is an important precursor in each type of pathway: glycine for purines and aspartate for pyrimidines. Glutamine again is the most important source of amino groups — in five different steps in the de novo pathways. Aspartate is also used as the source of an amino group in the purine pathways, in two steps.
103
What does de novo purine synthesis begin and end with?
De novo purine synthesis begins with PRPP (phosphoribosyl pyrophosphosphate) and ends with IMP (inosine monophosphate). The IMP is then derivatized into AMP of GMP.
This is regulated by feecdback inhibition, so if IMP, AMP or GMP is abundant, it inhibits this process.
104
What does the de novo synthesis of pyrimidines begin and end with?
De novo pyrimidine synthesis begins with L-Asp and the final product is UMP (uridine monophosphate). UMP can become CMP through transamination.
dTMP is made from dUMP and feedback inhibition from all the dNTPs makes sure all of them are synthesized.
