Lecture 8 - Glycogen, CAC and ETC

Question 1

How are PFK-1 and FBPase-1 regulates by energy state?

Answer 1

PFK-1:

1) High ATP/ ADP, ATP/AMP ratios and high citrate inhibit
2) low ATP/ADP and ATP/AMP ratios stimulate PFK-1

FBPase-1:

1) low ATP/AMP ratios inhibits FBPase-1

Note: FBPase-1 only has allosteric site for AMP not ADP

Question 2

What effect does F26BP have on PFK-1 and FBPase-1?

Answer 2

F26BP stimulates PFK-1 by binding to allosteric site, causes conformational change that increases affinity of PFK-1 for substrate (F6P)

Inhibits FBPase-1 by binding to allosteric site, causes conformational change that decreases affinity of FBPase-1 for substrate (F16BP)

Question 3

Describe how glucagon affects the activity of the bidirectional enzyme PFK-2/FBPase-2

Answer 3

Glucagon binds to GPCR, alpha subunit swaps GDP for GTP, dissociates from receptor, stimulates AC to make cAMP, cAMP binds to regulatory subunit of PKA causing catalytic subunits to dissociate and phosphorylate PFK-2/FBPase-2, causes the PFK-2 domain to be inactive and the FBPase-2 domain to be active, results in conversion of F26BP to F6P and decrease [F26BP], increases gluconeogenesis and decreases glycolysis

Question 4

Describe how insulin affects the activity of the bidirectional enzyme PFK-2/FBPase-2

Answer 4

Insulin stimulates phosphoprotein phosphatase which removes P from PFK-2/FBPase-2, causes FBPase-2 domain to be inactive and PFK-2 domain to be active, results in conversion of F6P to F26BP increasing [F26BP], increases glycolysis and decreases gluconeogenesis

Question 5

Glycogen synthesis requires __(1)__

Glycogen breakdown is __(2)__

Answer 5

(1) energy

(2) thermodynamically favorable, does not require energy

Question 6

Which organs primarily contain stores of glycogen?

Answer 6

Muscle, liver, kidneys

Question 7

Describe the glycosidic bonds glycogen

Answer 7

Branched molecule, linear regions linked by alpha 1–>4 and branches occur via alpha 1—>6 linkages

Question 8

How many reducing ends are there in glycogen? How many non reducing ends? Why is this structure important?

Answer 8

1 reducing end, many non reducing ends. Important b/c glycogen breakdown occurs at non reducing ends so having many non reducing ends means there are many points in the molecule where degradation can occur simultaneously

Question 9

What are the steps of glycogen synthesis? (Hint: there are 4)

Answer 9

1) Mutase converts G6P from glycolysis to G1P
2) G1P is activated by binding to UTP, this step results in release of pyrophosphate (PPi) which is readily hydrolyzed to 2 molecules of inorganic phosphate (Pi) which is highly exergonic and drives the reaction forward, results in UDP-activated glucose (1 P from G1P 1 P from UMP)
3) UDP-glucose is added to growing chain by glycogen synthase
4) When growing glycogen chain gets to long, branching enzyme cleaves off segment and reattaches to form an alpha 1–>6 branch

Question 10

What is the role of glycogen phosphorylase?

Answer 10

Performs opposite role of glycogen synthase, breaks down glycogen by breaking alpha 1→4 linkage via phosphorylization at non reducing end of chain thereby releasing glucose from glycogen chain and adds phosphate to it to form glucose 1 phosphate

Question 11

What is the ultimate product of glycogen breakdown in the muscle? Why is this helpful to the cell?

Answer 11

Glucose 6 phosphate, helpful b/c G6P can directly enter glycolysis and skips 1st step where hexokinase phosphorylates glucose, this allows the cell to spend 1 less ATP per glucose molecule that travels through glycolysis

Question 12

What is the ultimate product of glycogen breakdown in the liver? What enzyme allows this to happen? Why is this helpful/important?

Answer 12

G6P –> glucose via glucose 6 phosphatase. Important so that the glucose from liver can leave the liver cell and enter the blood stream to maintain blood glucose and glucose availability for other tissues instead of trapping it inside the cell for glycolysis in the liver

Question 13

How is glycogen synthesis and breakdown regulated? (term for regulation)

Answer 13

Reciprocal regulation: positively regulating (increasing activity of) one pathway while simultaneously negatively regulating (decreasing activity of) the other pathway.

Question 14

How is glycogen metabolism regulated allosterically?

Answer 14

1) G6P stimulates glycogen synthase and inhibits glycogen phosphorylase
2) AMP stimulates glycogen phosphorylase
3) ATP inhibits glycogen phosphorylase

Think of energy state: when energy demand is high, glycogen breakdown will occur, when energy demand is low, glycogen synthesis will occur

Question 15

How does glucagon regulate glycogen metabolism?

Answer 15

Glucagon binds to GPCR, alpha subunit swaps GDP for GTP and is activated, dissociates from GPCR, travels to AC and stimulates AC to make cAMP, cAMP binds to regulatory subunits of PKA causing catalytic subunits to dissociate, PKA will phosphorylate glycogen synthase to inactivate it. PKA will also phosphorylate phosphorylase kinase, which will phosphorylate glycogen phosphorylase, which will phosphorylate glycogen to release glucose 1P.

More generally: glucagon initiates a GPCR signaling cascade that results in phosphorylation activity by PKA that deactivates the glycogen synthesis pathway and activates the glycogen breakdown pathway.

Question 16

How does insulin regulate glycogen metabolism?

Answer 16

Insulin stimulates protein phosphatase 1 (PP1), which removes the phosphate from glycogen synthase and activates it so glycogen synthesis can occur. It also removes the phosphate from phosphorylase kinase and the phosphate from glycogen phosphorylase, inactivating those enzymes and inhibiting the glycogen breakdown pathway.

Question 17

In what types of tissue is the PPP active?

Answer 17

1) Tissue that is heavily involved in lipid synthesis (i.e. adipose tissue)
2) Active in tissue with rapidly dividing cells which need high quantities of RNA and DNA

Question 18

What are the 2 major products of the PPP?

Answer 18

1) NADPH (powerful reducing agent, necessary for reductive biosynthesis of fatty acids and cholesterol, important for antioxidant molecule synthesis)
2) Ribose 5 phosphate (precursor for ribose unit of nucleotide biosynthesis)

Question 19

What molecule from glycolysis feeds into PPP?

Answer 19

G6P

Question 20

What are the 2 main roles of the CAC?

Answer 20

1) produce high energy electron carriers that will be used in the ETC to produce ATP
2) It is a gateway to the metabolism of any molecule that can be converted to acetyl-CoA (Glucose, Fatty acids, Amino acids) and it produces OAA for gluconeogenesis and precursors for AAs

Question 21

Describe the reaction of pyruvate with PDH to form acetyl coA.

What is the role of this reaction with respect to the CAC?

Answer 21

pyruvate + coA-SH + NAD+ + coenzymes –> Acetyl-coA + NADH + CO2

Note: coenzymes = TPP, lipoate, and FAD

This is the transition phase between glycolysis and the CAC

Question 22

How is the PDH complex regulated?

Answer 22

Inhibited by: NADH, ATP, acetyl coA
(i.e. when energy supply is high, PDH is inhibited)
Activated by: NAD+, AMP and CoA
(i.e. when energy demand is high)

Question 23

What 4 enzymes in the CAC are points of regulation?

Answer 23

PDH, citrate synthase, isocitrate dehydrogenase, and alpha ketoglutarate dehydrogenase

Question 24

For citrate synthase, isocitrate dehydrogenase and alpha ketoglutarate dehydrogenase, what are the 4 means by which these enzymes are regulated?

Answer 24

1) substrate availability
2) Product inhibition by NADH
3) Allosteric activation by ADP
4) Competitive feedback inhibition (i.e. succinyl coA competes with acetyl coA for citrate synthesis)

Question 25

Why are the rates of glycolysis and the CAC integrated?

Answer 25

Because glycolysis produces pyruvate which feeds into the CAC so the rate of glycolysis determines the rate of the CAC. Additionally, the [citrate] from the CAC directly inhibits PFK-1 in glycolysis controlling the flux through glycolysis and thus through the CAC.

Question 26

Why is the CAC considered an amphibolic pathway?

Answer 26

Because it produces intermediates via the oxidation of pyruvate (catabolic) but it also has its intermediates siphoned off for other processes (anabolic)

Question 27

What 3 reactions drain intermediates of the CAC?

Answer 27

1) Glucose biosynthesis (OAA can be converted to malate and shuttled out of mitochondria to cytoplasm where it is converted back to pyruvate for gluconeogenesis)
2) alpha keto-glutarate and OAA can be starting materials for amino acid synthesis
3) Fatty acid synthesis (Acetyl coA is converted to citrate, shuttled out of the mitochondria into the cytoplasm, and converted to fatty acids in the cytoplasm)

Question 28

How can the rate of the CAC be increased?

Answer 28

By increasing the [intermediates]

1) Pyruvate –> OAA by pyruvate carboxylase
2) Odd chain FAs can be degraded to produce succinylcholine coA
3) AAs can be degraded to yield OAA and aKG

Question 29

What is the reaction catalyzed by pyruvate carboxylase?

Answer 29

Pyruvate + CO2 + ATP + H2O –> OAA + ADP + Pi

Question 30

How does acetyl coA affect the function of PDH? Of pyruvate carboxylase?

Answer 30

Acetyl coA inhibits PDH (when [acetyl coA] is high CAC is slowed/stopped)
Acetyl coA stimulates pyruvate carboxylase (when [acetylcoA] is high, pyruvate is converted to OAA and used to replenish CAC [OAA] or shuttled out of mitochondria for gluconeogenesis)

Question 31

In terms of oxidation, what is the purpose of glycolysis and CAC?

Answer 31

Oxidation of fuels to CO2 which results in the release of reduced cofactors NADH and FADH2

Question 32

In terms of oxidation, what is the purpose of the ETC?

Answer 32

Exergonic reoxidation of NADH and FADH2 releases free energy which is harvested to synthesize ATP by oxidative phosphorylation

Question 33

What is the standard reduction potential?

Answer 33

The affinity of a substance for electrons, thus higher SRP means that the substance will have a greater tendency to accept electrons and become reduced

Question 34

How can SRP be used to determine how electrons will flow in a reaction?

Answer 34

From lowest SRP to highest SRP

Question 35

Describe the malate - aspartate shuttle

Answer 35

NADH from glycolysis needs to be shuttled into the mitochondrial matrix to be used by ETC. NADH transfers its e- to OAA in cytoplasm, which is then converted to malate. Malate is shuttled across inner membrane to matrix where it donates e- back to NAD+ to produce NADH and OAA.

Question 36

How does reduction potential relate to the ETC?

Answer 36

The reduction potential of successive complexes in ETC increases with reduction potential of O2 being the highest so that e- keep flowing through the ETC

Question 37

Describe what happens in complex I of ETC

Answer 37

NADH transfers e- to redox centers of complex (Fe-S containing compounds) which then transfer e- to coenzyme Q to produce QH2. In this process, 4H+ are pumped into IMS from matrix.

Question 38

Describe what happens in complex II of ETC

Answer 38

Succinate is oxidized to fumarate by succinate dehydrogenase and 1 molecule of FADH2 is produced in complex II. FADH2 then passes e- to Fe-S redox centers of complex II, which then pass e- to coenzyme Q to form another molecule of QH2. No protons are pumped into the IMS by this action.

Question 39

Describe what happens in complex III of ETC.

Answer 39

2 molecules of QH2 transfer their 4 electrons to 4 molecules of cytochrome c (oxidized) to produce 4 molecules of cytochrome c (reduced). 4H+ are pumped into IMS in the process.

Question 40

Describe what happens in complex IV of ETC.

Answer 40

Each cytochrome molecules transfer their e- to redox centers of complex IV. Electrons are then finally passed to O2 (1/2 O2 + 2H+ + 2e- –> H2O). Thus, 2 molecules of H2O are produced in total. For each molecule of H2O produced, 2H+ are pumped into IMS.

Question 41

In the ETC, moving protons from matrix to IMS moves the protons against their concentration gradient. This requires energy. Where does that energy come from?

Answer 41

As e- are moved through the complexes, free energy released from flow of e- from low to high reduction potential pumps H+ through the complexes and from the matrix to the IMS

Question 42

What is complex V?

Answer 42

ATP Synthase

Question 43

What are the 2 subunits of ATP synthase? What are their general functions?

Answer 43

F1 (phosphorylation of ADP to ATP) and F0 (allows protons to flow from IMS to matrix)

Question 44

Describe how the F0 subunit works

Answer 44

F0 subunit is made of 1 a subunit, 2 b subunits and 12 c subunits. First, 1 proton travels through the a subunit and binds to an adjacent c subunit in IMS, then that causes the c subunit ring to rotate away from the entry point. As the ring rotates, a H+ is ejected from the next c subunit such that the H+ enters the matrix and the newly empty c subunit now aligns with the a subunit to accept another H+.

For each full rotation, 12H+ are pumped into matrix

Question 45

Describe how the F1 subunit works (binding change mechanism)

Answer 45

The F1 subunit is made of 3alpha subunits, 3 beta subunits and 1 gamma subunit that goes through the center of the F1. The beta subunits are the catalytic sites. The beta subunits can be in the “loose” (ADP+Pi bound), “tight” (ATP bound) or “open” (empty) conformations.

First, ADP + Pi bind to beta subunit and it takes on the loose conformation. Then, the gamma subunit rotates 120 degrees which causes that same beta subunit to phosphorylate ADP to produce ATP and now be in the tight conformation. The gamma subunit rotates another 120 degrees which causes the same beta subunit to now release ATP and be in the open conformation.

Question 46

How many ATP are produced by 1 full turn of the F0 subunit?

Answer 46

3 ATP
(1 ATP = 4H+ pumped back into matrix)

Question 47

How is the production of ATP by ATP synthase dependent on the proton motive force

Answer 47

Energy to drive ATP synthase is from the proton electrochemical gradient, protons moving down concentration gradient provides energy for ATP synthase to phosphorylate ADP

Question 48

How are electron transport and ATP synthesis are coupled?

Answer 48

Electron flow generates proton gradient that drives ATP synthesis

Question 49

What is an uncoupler?

Answer 49

An uncoupler destroys the proton gradient so that there is no electrochemical gradient to power the production of ATP. However, it does not stop the flow of electrons. Thus, uncouplers cause a flat line in the amount of ATP synthesized but show the consumption of oxygen continuing to rise. In this process, metabolic fuels are continually oxidized and electrons are moved through the ETC but all the energy that is generated is released as heat.

Question 50

What does thermogenin do?

Answer 50

Thermogenin is a natural uncoupler found in brown fat. It is a proton channel between the inner membrane and the matrix that provides an alternate pathway for H+ to take. Thus, it dissipates the proton gradient to some extent so it reduces the amount of ATP that can be produced. The difference between the amount of energy normally produced by ETC and the amount of energy turned into ATP is released as heat.

Uses FAs