Lecture #12 - Glycolysis and TCA

Question 1

Glycolysis (Definition)

Answer 1

Glycolysis – Anaerobic breakdown of glucose (6C) into 2 molecules of Pyruvate (3C)
- Glycolysis occurs in the cytoplasm
- Glycolysis is found in ALL organisms and in ALL cells
- Glycolysis is perterubed in many tumors and by many pathogens (Ex. PET Scans)
- Glycolysis is highly regulated

Glycolysis and TCA are the central carbon pathways to make ATP

Question 2

Gluconeogensis

Answer 2

Gluconeogensis – production of glucose from non-carbohydtrate precusors

Glycolysis is reciprically regulated with gluconegnesis (If glycolysis is high gluconeogensis would be low)

Question 3

Four major and general ways to regulate/modulate metabolic pathways

Answer 3

4 universal mechansims to regulate any given metabolic pathway (APPLIES to all metabolic pathways in all systems and ALL pathways ; use the same mechanisms to increase/decrease the rate of the pathway):
1. Substrate avaialibity
2. Allosteric Activation/Inhibition (reversible)
3. Covalent modification of enzyme (post translation modifications)
- Example - Phosphorylation of key enzymes status affects the rate that enzyme will catalyze reaction –> affects the rate of the pathway
4. Upregulation/downregualtion of genes that encode enzymes to Incerase/decrease synthesis of enzyme (long term control ; changes amount of enzymes in cell)

Question 4

Four major and general ways to regulate/modulate metabolic pathways - Substrate avaialibity

Answer 4

Control the amount of substrate that is avaible to the entire pathway
- Example – Glyclysis is regulated based on the availability of glucose –> regulate glycolysis by substrate availability using GLUT transporters

Question 5

Four major and general ways to regulate/modulate metabolic pathways - Allosteric Activation/Inhibition (reversible)

Answer 5

Allosteic activation is how a cell knows when to speed up/slow down a metabolic pathway (metabolic logic of cell)
- Uses Allosteric control of an enzyme in the pathway
- Allosteric modulators that are sensed by cell are often energy currency of cell or major end product of reactions (NADH/NAD+, ATP/AMP, Acetyl-CoA, or Fatty acids) (applies to all processes)

Only have allosteric activation/inhibition on key enzymes that are catylyzing key regulatable steps in the pathway

Question 6

Common Allosteric modulators

Answer 6

Allosteric modulators are:
1. Product of the enzyme
2. Energy currencey (ATP/AMP or NADH/NAD+)
- High energy (high ATP) –> ATP is an allosetric inhibtor (bind to and inhibit enzymes and slow reaction)
- Low energy (AMP is high ; ATP is low) –> AMP is the allosteric activator (binds to and activate enzymes and speeds up reaction)

Question 7

Ways to get glucose into cell

Answer 7

Two class of Glucose transoorters that mediate Glucose upatake:
1. Na Glcuose Co-transporter (SGLT2)
- In the kidney and Gut (Apical side)
- Absorbs glucose from the lumen of the intestine into the cell
2. GLUT4 (Muscle and Adpiose tissue)
- Facilitated glucose transporters
3. GLUT2
- In Gut (basal side)
- Puts glucose into blood
- Facilitated glucose transporters

Different glucose transports have different properties

Question 8

Facilitated glucose transporters

Answer 8

1. GLUT 1 – basal glucose uptake
   
   • Expressed in all cells
   • Low Km that is below the physiological concetration of glucose (high affinity for glucose) –> MEANS GLUT1 is always saturated –> Because it is always saturated it can’t be regulated
2. GLUT 2 (Liver and panecratic beta cells)
   
   • High Km that is above the physiological concentration of glucose –> not always saturated –> can be regulated
   • GLUT2 = used as a glucosensor
3. GLUT 4 (Muscle and adipocytes)
   – Medium Km
   
   • Insulin responsive glucose uptake
   • ONLY trasnports glucose in response insulin (high glucose)

Question 9

Why can GLUT2 be a glucose sensor

Answer 9

Can be used as a glucose sensor because the rate of glucose transport is proportional to the amount of circulating glucose in the blood –> cell knows how much glucose is in the blood based on how much glucose goes into the cell
- Used in beta cells to know concetration of glucose in blood to know how much insulin to release

Works because GLUT2 has high Km –> MEAN GLUT2 has a big dynamic range –> allows the amount of sugar that goes into the cell to be proportional to the amount of sugar in circulation

NOTE - Glucokinase also functions as sensor –> GLUT2 and GK allows liver and pancreus cells to know glucose concentration

Question 10

Potencey of Insulin and Glucagon

Answer 10

Insulin and Glucagon are the most potent hormones in the body (antagonize each other)

Ex. Small amount of insulin can lower the blood glucose (don’t want to release too much insulin)
- Needs to know how much sugar in the body to secrete the right amount of insulin

Fasting – Glucagon is high Vs. Fed – insulin is high

Question 11

Phases of Glycolysis and regulatable steps

Answer 11

1. Prep phase –> conusmes 2 ATP to prep the sugar ready to be able to extract energy from it
2. Pay off phase –> make 2 NADH and get net 2 ATP increase

3 steps are regulated ; rest of steps are at equilibrium

Regulated steps:
Steps 1 - hexokinase/glucokinase –> uses hormone increase
- Insulin stimulates Glucokinase
Step 3 - PFK1
Step 10 - Pyrubate Kinase
- Uses Allosteric regulation + covalent modifcations

Question 12

Gibbs free energy (dG)

Answer 12

Gibbs Free energy (free energy) - amount of energy that is available to do work at constant pressure
- dG = difference in free energy between the reactants and the products
- dG is a measure of how spontaneous a reaction is (how likely it is to go forward)
- dG does not indicate how fast the reaction occur

More negative dG = more favorable a reaction is (more likely to move forward)

Question 13

What steps do you regulate in a pathway

Answer 13

Regulate steps where dG is VERY negative because they are very spontenous
- When dG is very negative the reaction wants to go fowards (reaction is almost irreverisble)

Want to regulate irreversible steps because once you go foward the energtic barrier to go backwards is too high (only want do the step if you are sure you will not want to go back)

IN Example - regulate steps 1, 3, 10

Question 14

Regulated steps in glycolysis

Answer 14

Regulation happens at steps where dG is very negative:
Step 1 - HK/GK
Step 3 - PFK-1
Step 10 - Pyruvate Kinase

Other parts of the pathway have +dG or are at equilborum (not regulated) –> go fowards or backwards depending on the amount if substrate/product

Question 15

First step of glycolsysis

Answer 15

When glucose goes into the cell is phosphorylated –> puts a negative charge on sugar = traps glucose in cell)
- Done by Hexokinase/Glucokinase
- Need to trap glucose in cell because glucose transports can move sugar in and out –> phosphorylating glucose prevenst it from leaving

Phosphorylation ALSO helps control the rate of glycolysis because Glucose-6-phoshate inhibits HK –> MEANS that phosphorylation helps control the rate HK and therefore the rate of glycolysis
- NOTE - hexokinases have a low Km

Question 16

Why do enzymes consume ATP when they catalyze reactions

Answer 16

Enzyme hydrlyze ATP when they catylzye reactions to harness the energy from ATP hydrlysis to drive forward an unfavorable reactions (couple an unfavorable reaction with a favorable reaction)

Example - Glucose –> Glucose-6-Phospahte) –> dG = +33 (react would not happen by itself) Vs. dG for ATP hydrolysis = -7.3
- END couple phosphorylation and ATP hydrlysis dG is -4.0 (favorable)

Question 17

Hexokinase Vs. Glucokinase

Answer 17

Hexokinase - low Km (lower than physiological concetration of glucose) –> it is easily saturated with glcuose
- Inhibited by glucose-6-phpshate
- In most cells

Glucokinase – has a high Km (not easily saturated)
- In liver + pancreatic beta cells +brain
- NOT inhibited by Glucose-6-Phosphate
- High Km –> enzyme has big dynamic range –> allows Glucokinase to function as a glucosensor
- If had Low Km –> enzyme would be always be saturated –> can’t be regulated/can’t sense the glucose conctration

Question 18

Affect of Km on GK and HK sensing glucose changes

Answer 18

Chart - shows that HK is saturated at a low glucose concetration Vs. GK is not
- Difference in Km for Glucokinase V.s hexokinase makes glucokinase more sensative to changes in the concentration of glucose in the cell
- Rate of glucokinase changes based on teh glucose ceonctartion BUT the rate of hexokinase does not

Question 19

Ways that HK and GK are regulated

Answer 19

1. Km regulates GK
2. HK/GK are also regulated by substrate availability (Substrate availability is done using Glucose transports that are only at the cell surface when there is high glucose)

Question 20

Regulation of PFK1

Answer 20

Phosphofrutokinase 1 (PFK1) reaction – Fructose –6-phosphate –> Fructose 1,6- Bisphosphate

PFK1 is the KEY regulatory point in glycolysis

PFK-1 is regulated by
1. ATP/AMP ratio
2. Fructuose-2,6-Bisphosphate (allosteic modulator) (MAIN WAY)

Chart - PFK1 is sensative to amount of AMP –> When add AMP –> get increase in PFK-1 Activity (stays high)
- No AMP - Have peak at low ATP then decrease

Question 21

Fructose-2,6-bisphosphate regulation of PFK1

Answer 21

Fructose-2,6-BP promote glycolysis over gluconeogenesis

F-2,6-BP – activates kinase actaivity in PFK-1
- Increase in Fructose-2,6-BP –> promote glycolysis
F-2,6-BP – Inhibit FBPase-1
- Decrease in Fructose-2,6-BP –> activates phosphotase activity of PFK1 (FBPase1) promote gluconeogensis

Question 22

Effect of fructose-2,6-Bisphosphate on PFK-1

Answer 22

Fructose-2,6-Bisphosphate Activates PFK-1 by lowering Km for PFK-1 and increasing the Km for FBPase-1

PFK1 - has kinase activity AND phosphatase activity –> Fructose-2,6-Bisphosphate Activates the kinase and inhibits the phosphatase of PFK-1
- Phosphatase = involved in gluconeogensis (Fructose-2,6-Bisphosphate inhibites gluconeogensis)

Question 23

What controls the amount of fructose-2,6-Bisphosphate- Fasting

Answer 23

Uses PFK2 (makes Fructose-2,6-Bisphosphate)

Fasting (low glucose) –> release glucagon –> Glucagon binds to GPCR –> cAMP is increase –> cAMP activates PKA –> PKA phosphorylates and activates the phosphatase domain of PFK-2 AND phosphorylates and inactivates Kinase domain of PFK2 –> PFK-2 dephosphporylates Fructose-2,6-Bisphosphate –> get decrease in fructose-2,6-Bisphosphate –> decrease in the allosetric actaivtor of PFK-1 –> glycolysis decreases ; gluconeogensis increase
- Don’t want to break glucose in starvation

Question 24

What controls the amount of fructose-2,6-Bisphosphate- Fed

Answer 24

When eat (high glucose)–> insulin is released –> leads to activation of a phosphatase –> phosphatase dephosphorylate and inactiavtes the phosphatase domain of PFK2 and dephosphortlatse and actiavtes kinase domain of PFK2 –> get increase in Fructose-2,6-Bisphosphate –> fructose-2,6-Bisphosphate actaivtes the kinase actaivity of PFK-1 –> increases the rate of glycolysis and decrease gluconeogensis
- High glucose = want to break down glucose to make ATP

NOTE - insulin stimulated phosphotase removes phosphate from PF and PFK2-/FBPase2 –> promoves glycolysis over gluconeosgesis

Question 25

Insulin and PFK1

Answer 25

PFK1 = regulated by insulin and glucagon because they will inhibit or activate PFK2 which affects the level of Fructose-2,6-bisphosphate that is made which affects PFK1 activity

Question 26

Regulation of Pyruvate Kinase

Answer 26

PK Reaction - Phosphopyruvate --> Pyruvate + ATP PK is regulated using 4 general mechanisms 1. PK regulated by phosphorylation (post translational modication) 2. Transcription of Pyrivate kinase genes is inhibited by cAMP and activated by Insulin - Activity of PK depends on Insulin Vs. Glucagon 3. Allosteric activation/inhibition - High ATP = inhibits PK ; Low ATP (high AMP)= activates PK

Question 27

Glucagon Vs. Insulin Effect on PK

Answer 27

Starvation - Glucagon --> binds to GPCR --> get increase in cAMP --> cAMP activates PKA --> PKA phosphorylates and deactivates pyruvate kinase Fed - Insulin increases --> insulin increases a phosphatase --> Pyruvate kinase is not phosphorated --> PK is activated SHOWs PTM is a key mechanism to regulate metabolic enzyme

Question 28

Importance of the discovery of insulin

Answer 28

Discovered by struggling surgeon who never work in lab Discovery of insulin made diabetes a manageable disease (before T1D was a death sentence)

Question 29

Oxygen in Glucose metabolism

Answer 29

Glycolysis does NOT require oxygen BUT rate of glycolysis is affected by the presence or absence of oxygen (Pasture effect) Decrease in O2 = increase the rate of Glycolysis ; Increase in O2 = decrease rate of Glycolysis WHY – because mitocondria need oxygen to do OxPhos --> No O2 --> Mit don't work (not making ATP) --> need faster rate of glycolysis to make enough ATP

Question 30

Why can glyclysis slow down in high O2

Answer 30

Have oxygen--> slow down glycolysis because can get ATP using mitochondria (don’t need glycolysis to go fast to make a ton of ATP) Complete oxygenation of glucose (in mitocondria) gives 38 ATP Vs. Glycolysis gives 2 ATP - When have oxygen prefer to use mitocnidria to make ATP = don’t relay on glycolysis to make ATP = glycolysis can be slower

Question 31

What happens to glycolytic intermediates in low O2

Answer 31

IN experiment – O2 decrease --> amount of glycolytic intermediates increases --> means glycolysis is faster when O2 decreases

Question 32

Affect of AMP and O2 on Glycolysis rate

Answer 32

System is very sensitive to AMP level - Anaerobic (20% increase in AMP) --> get 30X increase in Gyclysis rate AMP (high in low O2) – increases Pyruvate kinase and PFK-1 --> Low O2 increases the rate of glycolysis by causing AMP to activate enzymes in glycolysis AND because AMP inhibites FBPase1 - FBPase 1 = decreases glyclysis = inhibiting inhibitor of glycolysis

Question 33

Warburg Effect

Answer 33

Warburg effect – shift in energy production from oxidative phosphorylation to glycolysis in the presence of oxygen (contrasts Pasterure effect) Seen in cancer - Cancer have functioning mitochondria BUT they cells don’t use Oxidative Phosphorylation to make ATP INSTEAD they increase the rate of and use glycolysis at the main ATP producing pathway - Weird because get more ATP when completely oxidize glucose (use ETC and OxPhos)

Question 34

Where can you see the Warberg Effect

Answer 34

See in cell culture --> media becomes acidic (changes color) because cells do a lot of glycolysis --> make a lot of pyrvuate --> pyruvate can be reduced to Lactate (secreted by cancer cell) - Need to make lactate to make more NAD+ (IF only did glyclysis then would run out of NAD+ --> to get more NAD+ reduce pyruvate to lactate) Warburg = reason PET scans work (because tumors increase rate of glycolysis = light up when give person labeled glucose)

Question 35

NADH going into mitocondria

Answer 35

NADH (made in glycolysis) can’t cross the inner mitocindral membrane to be used in TCA cycle Solution - two shuttle systems shuttle electrons from NADH to mitocondria: 1. Glycerol 3-phosphate Shuttle 2. Malate-Aspartate Shuttle Shuttle systesm will: 1. Transfer NADH (reducing equivilent) to the mitocondira 2. Generate NAD+ (NAD+ is used to keep glycolysis going under aerobic conditions)

Question 36

What happens to the pyruvate at the end of glycolysis

Answer 36

After glycolysis pyruvate needs to make a decision - it can: 1. Go to the mitochondria to be fully oxidized (TCA) 2. Be converted to glucose in gluconeogensis (done in fasting) 3. Get reduced to Lactic Acid and secreted from the cell (Done when have no O2) The metabolic fate of pyruvate is determined by the cell type and the metabolic state (charge energy) of the cell

Question 37

Fate of Glucose-6-Phosphate

Answer 37

Glucose in cells can go through glycolysis OR can go to the pentose phosphate pathway (PPP) - Glu-6-phosphate can be oxidized and go to PPP PPP is regulated in the same way as glycolysis - First step of PPP = Glu-6-phosphate dehydrogenase (commitment step/step that is regulated)

Question 38

Function of PPP

Answer 38

1. Makes 5 carbon ribose ( used to make nucelic acids) - Need nucleotides for DNA to replicate --> need 5 carbon ribose to make make nucleotides 2. PPP makes tons of NADPH - NADPH regernates glutathione (glutathione is defense against oxidative stress) - NADPH = needed for reductive biosynthesis and to do redox to repair oxidative damage Vs. NADH is used for energy

Question 39

TCA - Overview

Answer 39

For TCA to happen - Pyruvate needs to go to mitocondria 3 Steps of TCA are regulated (ALL have -dG ; are irreversible) - Regulated by 4 main mechanisms

Question 40

Steps that are regulated in TCA

Answer 40

PDH (NOT TCA cycle yet) 3 Steps are regulated: 1. Citrate synthase 2. Isocitrate dehydrogenase 3. Alpha ketoglutarate dehydrogenase

Question 41

Pyruvate dehydrogenase

Answer 41

PDH = NOT in TCA (right before) Before Pyruvate can go to the TCA cycle it needs to pass through Pyruvate dehydrgenase (gatekeeper) - PDH reaction - Pyruvate --> NADH + Acyyl-CoA + CO2 (Acytl-CoA will go to the TCA) - PDH = regulated - Happens in mitocondria - PDH links glycolysis to TCA

Question 42

Citrate synthase

Answer 42

Reaction (condensation reaction) - Actyl-CoA + Oxoloacteate --> citrate - Marks the enrty of Acytl-CoA into the TCA cycle - REGULATED Step (-dG ; favorable reaction)

Question 43

Isocitrate dehydrogenase

Answer 43

Reaction – Isocitrate --> alpha-ketoglutarate - REGULATED Step (-dG) Have Two forms of isocitrate dehydrigenase: 1. uses NAD+ (In mitocondira - used in TCA) 2. Uses NADP+ (in mitocondria and cytosol)

Question 44

Alpha-ketoglutarate dehydrogenase

Answer 44

Reaction – Alpha-ketoglutarate --> Succinyl-CoA - REGULATED Step (-dG) - Enzume is similar to PDH

Question 45

Succinyl-CoA synthetase

Answer 45

Succinyl-CoA synthetase - Succinyl-CoA -> Succinate + GTP/ATP - Most of the energy released is used to drive GTP/ATP = low net dG Succinyl-CoA synthetase makes GTP BUT can Nucleoside Diphopshate Kinase converts GTP to ATP - GTP and ATP are energetically equivilent = no change in free energy - Nucleoside Diphopshate Kinase is at equilibrium (dG = 0)

Question 46

REST of reactions in TCA (HE SKIPPED)

Answer 46

ALL NOT REGULATED Succinate Dehydrigenase - Succinate --> Fumarate - Only membrane-bound enzyme in the TCA Cycle (on inner mitochondrial membrane) Fumarase - Fumarate --> L-Malate Malate Dehydrigenase - L-Malate --> Oxoloacetate - large +dG = favors L-Malate formation BUT Citrate Synthase is highly exergonic (ΔG’0 = -7.7) --> pulls this reaction toward oxaloacetate formation

Question 47

End result of TCA + glycolysis

Answer 47

Get 3 ATP per NADH and 2 ATP per FADH2 TCA (4 oxidation reaction to fully oxidize glucose) - 6 NADH + 2 FADH2 + 2 ATP --> makes 24 ATP total per glucose TOTAL (glyclyiss and TCA) --> makes 38 ATP per 1 glucose

Question 48

Major roles of TCA cycle

Answer 48

1. Generate energy (ATP and NADH) - Main energy producing pathway in the mitochondria matrix - NADH is used to make energy for OxPhos 2. Provide substrates (or intermediates) for biosynthetic processes - Intermdiates that are needed for reductive biosynthesis of other molecules - Get intermediates to make make Fatty acids and sterols + heme + glutamte (Amino Acid) + purines + pyridmines

Question 49

Substrates made by TCA

Answer 49

Example 1 – RBCs have hemoglobin (requires heme to bind to O2) - Intermediate that is needed to make heme is Succinoyl coA --> Succinoyl-CoA goes from TCA to make porfilin --> make heme --> used in RBCs Example 2 – Alpha Keto-glutarate - Alpha-ketoglutarate – makes glutamate --> glutamate can be used for other things (ex. Purine biosynthesis) Example 3 – Citrate can leave TCA cycle and be used to make fatty acid/sterol - IF ingest fats get fatty acids OR you ingest sugares --> get citrate and make fats

Question 50

Summary

Answer 50

To know for test: 1 – 4 universal regulations 2 – Major functions of TCA 3 – Role of PPP 4 – Know fates of Pyrvate once it is made (can be fully oxidaes in TCA in mitocondira or converted to glucose in fasting conditions in gluconeogesis or can be reduces to lactate when have no oxyegns)