BCH210 - Lecture 18 - Carbohydrate metabolism part 2

glycogen synthesis steps

step 1a: glucose is phosphorylated by hexokinase to G-6-P → this decides the fate of what happens to glucose in that cell

step 1b: G6P is isomerized by phosphoglucomutase to form G1P

step 2: UDP-glucose pyrophosphorylase uses UTP to activate G1P to make UDP-glucose

step 3: Glycogen synthase adds UDP-glucose, to the non-reducing ends of glycogen creating a new α1,4 glycosidic bond (you add UDP-glucose onto the glycogen chain, building the glycogen molecule)

glycogen

-excess glucose can be stored as it (anabolic pathway) → when we eat a meal (fed state)

-its stored specifically in muscle cells and liver tissue

-its a key branched glucose polymer

if hexokinase is turned on what must happen?

-it must be used by the cell

what happens when glucose is phosphorylated to G6P?

-it can’t move across the membrane using the GLUT transports, so G6P is instead used to make glycogen

isomerize reaction

-converting the molecule to an isomer of itself

phosphoglucomutase reaction

1. insulin signalling

2. glucose uptake and phosphorylation

3. the -OH on G6P attacks the phosphorylated serine (taking the phosphate)

4. it then becomes an intermediate; glucose 1,6-bisphosphate

5. the serine then attack the phosphate on C6 of glucose 1,6-bisphosphate (taking the phosphate)

6. glucose 1,6-bisphosphate then becomes G1P + UTP

7. → UDP-glucose → glycogen synthesis

**then it enters the glycogen synthesis pathway to produce and store glycogen

what energy is used for glycogen synthesis

-UTP instead of ATP (uracil triphosphate)

why does Glycogen Synthase need UDP-glucose for glycogen synthesis? why can’t it just use Glucose 1-phosphate?

G1P + Glycogen (n) -> Glycogen (n+1) + Pi delta G knot > 0

-the energy in the phosphate bond is not enough to carry it over and activate to make

it into a favourable reacton (to have a negative change in the gibb's free energy) the hydrolysis ends up being a reversible reaction (its hard to make a molecule when you have a reversible reaction, where its gonna be made then unmade in the same response) so in order to overcome this we need to couple it with a different reaction, thermodynamically this has to be coupled with other enzymatic reactions

**use UTP to make the reaction more favourable

UDP-glucose pyrophosphorylase

-breaks the bond in UTP, removing one phosphate (uridine)

-then adds it onto G1P to make UDP-glucose

**makes overall reaction more favourable

→ G1P + UTP ⇌ UDP-G + PPi => ΔG_o = 0

PPi + H2O→ 2Pi => ΔG_o = -19 kJ/M

Glycogen synthase

-breaks the bond between UDP and glucose, the -OH on glycogen attacks that bond and binds to glucose forming an α1,4 glycosidic bond

-because this is an irreversible reaction, we need a separate enzymatic reaction to now break these bonds and break it apart since its not reversible

→G1P + UTP ⇌ UDP-G + PPi => ΔGo = 0

PPi + H2O→ 2Pi => ΔGo = -19 kJ/M

UDP-G + Glycogen _(n) → Glycogen _(n+1) + UDP

=> ΔGo = -13 kJ/M

glycogenolysis

-is a phosphorolysis reaction

steps of glycogenolysis

step 1a: Glycogen phosphorylase uses free Pi (reacts at the anomeric carbon) to create and release G1P from the non-reducing ends of glycogen (one at a time)

step 1b: G1P cannot be used directly in glycolysis and must first be converted to G6P by phosphoglucomutase

step 2(LIVER ONLY): G6P is dephosphorylated to Glucose and released back into the blood along its concentration gradient to maintain blood glucose levels (using G6-phosphatase)

**The muscles will use G1P to break it down and release energy and can only be used in the muscle no where else

glycogen is stored in the liver and muscle tissue. There is 300-500 g of glycogen in muscle tissue (depending on muscle mass), while can liver can store 80-100 g of glycogen.

which of the following is most CORRECT regarding the use of these carbohydrate stores to maintain homeostasis?

-liver glycogen is primarily used to maintain blood glucose homeostasis, due to the liver’s ability to release glucose back into the bloodstream

-only liver cells express G6P so only liver cells can convert G6P back to glucose, allowing it to now enter the bloodstream

where is glycogen found

-the liver and muscle tissues

how are glycogen levels regulated?

-balance of synthesis and degradation of glycogen is regulated by glucose availability and enzyme activity (epinephrine and glucagon) → stored when glucose is in excess, broken down when glucose is needed (there will be a net production of glycogen and store it for future use)

what regulates glycogenolysis?

-epinephrine and glucagon signaling regulates glycogenolysis

→when glucose is low, the glycogen will be broken down and added to the blood and maintain blood glucose levels

what is glycogen regulation key for?

-it is key to maintaining the levels of glycogen in all these tissues

what regulates glycogen synthesis?

-insulin signalling

what are the 2 ways insulin signaling regulate glycogen synthesis?

1. dephosphorylation and inactivation of glycogen phosphorylase(this breaks down glycogen, so you want it inhibited)

2. dephosphorylation and activation of glycogen synthase (builds up glycogen)

how do epinephrine and glucagon signaling regulate glycogenolysis?

-phosphorylation and activation of glycogen phosphorylase

-phosphorylation and inactivation of glycogen synthase

**signals activation of a kinase

what is glycogen phosphorylase allosterically inhibited by?

-Glycogen phosphorylase is also allosterically inhibited by ATP and G6P and stimulated by AMP (when there’s a lot of AMP)

what does the liver not use for energy?

-the liver doesn’t use glucose for energy

what does the muscle tissue do to G6P?

-it brings into glycolysis and breaks it down to produce ATP, CO2 and lactate

**the glycogen and glucose can never leave once in the cell and will only be used for muscle tissue

if muscle cells contain Glycogen, a form of carbohydrates in animal cells, why does the meat we may consume not contain any carbohydrates?

-glycogen is used up by the muscle cells, resulting in a loss of carbohydrates in meat

-when an animal dies, that stops blood flow, which is how oxygen is transported, so it stops oxygen transport to those tissues and stops glucose flowing and fuels flowing within the cells and those cells don't die quickly, they instead use all their tissue and resources to survive for as long as possible, its only when they exhaust all their fuel sources that they will undergo cell death

-so the muscle cells being starved of glucose and oxygen, start to use their internal resources, glucose levels drop which signal the breakdown of that glycogen as that glycogen is broken down its used to maintain homeostasis for as long as possible

**liver cells do not exhaust all their carbohydrates

anerobic glycolysis

-no mitochondria (ex.RBC) or no O₂

→ the need to regenerate NAD+

-this is where pyruvate gets converted to lactate using NADH (which allows glycolysis to still occur)

what molecule helps glycolysis keep going?

-NAD+

→ without it glycolysis can’t keep going

aerobic metabolism

-mitochondria + O₂ available

→the pyruvate can enter into the mitochondria

what does the production of lactate indicate?

-becomes an indicator of hypoxia within the tissues where cells might be deprived of oxygen

what else must happen for glycolysis to occur?

-pyruvate must go somewhere

what happens when pyruvate builds up?

-this turns off the glycolytic pathway and pyruvate is converted into lactate

which is faster ATP breakdown or ATP synthesis?

-ATP breakdown is faster than ATP synthesis

metabolic acidosis

-results in the breakdown of ATP, which changes the pH in our blood causing this

-lactate is present during acidosis but its not the cause of it

why is lactate not the cause of acidosis?

-because it is never in its acidic form, its always deprotonated, it can’t be protonated (pKa = 3.8), so it can’t donate a proton to contribute to acidosis

what does the breakdown of ATP generate?

-free protons

what happens if lactate builds up?

-if lactate builds up, lactate dehydrogenase will turn off, through product inhibition

what is the process of pyruvate to lactate?

1. pyruvate (3C) comes from glycolysis, but there’s no mitochondria or O2 present

2. lactate dehydrogenase (LDH) catalyzes this reaction, using NADH.

3. NADH is oxidized to NAD+ (NAD+ is regenerated, and allows for glycolysis to continue)

4. lactate is now formed (3C

what are the 4 main fuels seen in cells?

1. ATP (not stored generated as needed)

2. creatine phosphate

3. carbohydrates

4. fats (one of the biggest fuel tanks)

what can fat do?

-it can generate a lot of energy but its a slow process, not generated quickly.

what can glycolysis and carbohydrate metabolism do?

-they can generate a little energy but its done way faster

mitochondria

-a double membrane organelle

what does the pyruvate do once its out of glycolysis?

-the pyruvate can now move from the cytoplasm where

glycolysis is occuring and move into the mitochondria

where it can be broken down or oxidized in the TCA cycle

and then generating energy in the ETC

mitochondria- inner mitochondrial membrane

-very impermeable, things can’t move through it very easily

-allows it to create conc. gradients

mitochondria - matrix/lumen

-where all the enzymes are present for al the enzymes involved in few metabolism

mitochondria- permeable membrane

-allows molecule to move in and out of the membrane

pyruvate dehydrogenase complex (PDC)

-connects glycolysis and the CAC cycle

-if this enzyme turns off these 2 pathways are disconnected and separated, so very vital step

-links the production of pyruvate in cytoplasm with the TCA Cycle in the mitochondrial matrix

-it is a large complex of multiple copies of 3 different enzymatic subunits

-moves through porons to get to that intermembrane space in the mitochondria and then it needs to be converted into something useful

what are the 5 cofactor for catalysis that PDC requires?

1. thiamine pyrophosphate (TPP) → Vitamin B₁

2. lipoamide

3. FAD → Vitamin B₂

4. Coenzyme A → Vitamin B₅

5. NAD+ → Vitamin B₃

**almost all B vitamins you consume have required roles within energy metabolism

prosthetic group

-a cofactor that is permanently or covalently attached to that enzyme (it won't be released)

coenzymes

-these are cofactors that can be released, they interact with the enzyme and then can leave to the next reaction

which out of the 5 cofactors for PDC are prosthetic groups? (3)

1. TPP

2. lipoamide

3. FAD

which out of the 5 cofactors for PDC are coenzymes? (2)

1. coenzyme A

2. NAD+

what is an advantage of the enzyme subunits forming a complex?

-having these processes occurring within close proximity to eachother, either localizing these enzymes within a single organelle or bringing them together in a protein complex, makes these processes occur more efficiently and faster.

-this is an advantage because if a cell can generate energy faster or save energy by regulating this process easier that gives us an advantage in growth and survival within a complex environment

PDC reaction (pyruvate de-carboxyl reaction)

-3 enzymatic reactions in 1

-occurs in the mitochondrial matrix

-pyruvate (from the breakdown of carbohydrates) is oxidized and activated, forming Acetyl CoA

-this reaction is a redox decarboxylation reaction, producing CO₂ and NADH

-this reaction has a large, negativeΔGº´ (i.e. highly favourable/spontaneous) but doesn’t use ATP, all the cofactors participate because of how complex the process is

-is a 5-step reaction (consists of 3 enzymes coming together, E1, E2, and E3)

→ ΔGº´= - 33 kJ/mol (indicates its favourable and irreversible as it connects 2 pathways)

what about the PDC reaction contributes to its favourability?

-when a product of the reaction disappears it drives the reaction forward (since CO2 is released in this reaction, it disappears immediately (is used up)) so we see this as a potential driving force

-when a relatively unstable product of the reaction disappears it means that the reaction essentially becomes irreversible

E1 enzyme of PDC

-pyruvate dehydrogenase

E2 enzyme of PDC

-dihydrolipoyl transacetylase

E3 enzyme of PDC

-dihydrolipoyl dehydrogenase

process of PDC reaction

1. decarboxylation of pyruvate

2. oxidation of the resulting hydroxyethyl group producing NADH (all the cofactors contribute)

3. coenzyme A then binds onto it to form acetyl CoA

what is the driving force of the PDC reaction?

-CO2 (its oxidized and released)

acetyl CoA

-acetyl group = = a 2 carbon group that came from pyruvate bound to coenzyme A

what is PDC crucial for?

-linking 2 pathways together

where are the 2 places energy is captured in PDC reaction?

-energy is captured in 2 places : 1. within acetyl CoA and 2. NADH and they both provide energy that can be used to fuel the generation of ATP later on

what molecules bring feedback inhibition to PDC (its regulation since its favourable)?

-acetyl CoA inhibits E2 enzyme by either binding to the active site or through allosteric regulation

-NADH inhibits E3 enzyme by binding to it

-it will turn off the process and turn off the enzyme causing it to back up then pyruvate will start to build up and turn off glycolysis

what stimulates PDC?

-ADP and Ca2+

how else can PDC be controlled?

-by post-translational modifications (PTMs)

for E1:

→ an active E1 PDC is phosphorylated by PD kinase, which inhibits it and makes it inactive (using ATP as well)

→ an inactive E1 PDC is dephosphorylated by PD phosphatase, which stimulates it and makes it active (using H₂O)

what effect does high levels of acetyl CoA have on PDC, PD kinase and PD phosphatase?

PDC → inhibits

PD kinase → stimulates

PD phosphatase →inhibits

what effect does high levels of NADH have on PDC, PD kinase and PD phosphatase?

PDC →inhibits

PD kinase →stimulates

PD phosphatase → inhibits

what effect does high levels of pyruvate have on PDC, PD kinase and PD phosphatase?

PDC →stimulates

PD kinase →inhibits

PD phosphatase →stimulates

what effect does high levels of ADP have on PDC, PD kinase and PD phosphatase?

PDC →stimulates

PD kinase →inhibits

PD phosphatase →stimulates

what effect does high levels of Ca2+ have on PDC, PD kinase and PD phosphatase?

PDC →stimulates

PD kinase →inhibits

PD phosphatase →stimulates

what are the products of PDC reaction?

-the products are 2 CO2, 2 NADH, and 2 acetyl CoA (occurs twice since 2 pyruvates are produced from glycolysis)

→ pyruvate + NAD+ + CoA → acetyl-CoA + NADH + CO₂

muscles at rest (high energy state)

-NADH and Acetyl CoA (products) (high levels of them) stimulate the kinase to inhibit the complex by phosphorylation

**E1 is important to inhibit since its a decarboxylation step

muscles exercising (low energy state)

-lots of glycolysis has started and pyruvate is building up

-pyruvate and ADP levels are high, so this turns on the complex and all these processes converge to ramp up production of acetyl CoA from E1

**as the concentrations increase the activity will increase proportionately

allosteric control of PDC

-Allosteric control of PD kinase by Acetyl CoA, NADH, pyruvate and ADP causing conformational change

what molecule is at the center of energy metabolism

-acetyl-CoA

→all the different metabolic fuels can generate acetyl-CoA

which molecule is a very high energy molecule?

-ethanol

the Citric Acid Cycle (CAC or TCA or Kreb’s cycle)

-oxidizing fuel

-links the breakdown of fuel molecules to ATP production in Oxidative Phosphorylation

-has 2 phases: phase 1 - breakdown fuel, phase 2 - generate ATP

-allows molecules to now go into the oxidative phosphorylation in the ETC once the CAC process is done

-is the hub of mitochondrial oxidation and uses acetyl CoA supplied by breakdown of glucose, fatty acids and amino acids

-the overall pathway is favourable despite the positive standard free energy of Malate Dehydrogenase (MDH)

-is considered an amphibolic pathway linking anabolic and catabolic pathways via Acetyl CoA and TCA intermediates

what must happen for acetyl CoA to be used for energy?

-for it to be used for energy it enters the CAC

what is the driving force of the CAC cycle?

-CO2 is the driving force that is pushing this reaction forward

what is phase 1 of CAC?

-breakdown fuels

-fuels are oxidized to CO2, generating NADH and FADH2

-the oxidation of fuels

what is phase 2 of CAC?

-generate ATP

-this is all about that NADH and FADH2

-this is where all the oxygen comes in

-ATP generation from oxidative phosphorylation

what is stage 1 of CAC?

-oxidative carboxylation

→acetyl-CoA (2C) combines with oxaloacetate (4C) to produce citrate (6C) – this starts the CAC cycle

→ during the cycle, 2Cs are oxidized to CO2, regenerating oxaloacetate in the process (releases CO2 driving the reaction forward and the NADH ends up in the ETC)

what is stage 2 of the CAC?

-regenerate oxaloacetate

-during the cycle, 2Cs are oxidized to CO2, regenerating oxaloacetate in the process (releases CO2 driving the reaction forward and the NADH ends up in the ETC)

-we also lose 2 carbons

succinate dehydrogenase

-is a membrane-bound protein linking the TCA cycle to oxidative phosphorylation and the ETC

-has a dual function

-helps succinate turn to fumarate

-directly involved in ETC (SDH (Complex 2) + Q)

what are the key notes of the products made in CAC?

-the GTP is made by substrate level phosphorylation → (it has the same amount of energy as ATP and we can consider them identical)

-2 H2O molecules are needed by reactions for citrate synthase (to turn acetyl CoA + OAA to citrate) and fumarase (to turn fumarate into malate)

-4 pairs of electrons are used to reduce and form 3 NADH and 1 FADH2 for oxidative phosphorylation to fuel the ETC

what type of reactions are the steps that need to be regulated?

-favourable reactions are the steps that are regulated

what are the regulated steps in CAC?

-steps 1, 3, and 4

where does CO2 carbons come from?

-come from oxaloacetate (in 1 round)

which TCA intermediates can be made from an amino acid?

-OAA, citrate, malate, succinyl CoA, and a-ketoglutarate

how is the CAC regulated?

-low energy and calcium stimulate the cycle

-high energy, reduced coenzymes or products inhibit the cycle

what regulates mitochondrial bioenergetics?

-allosteric and/or covalent modification of the PDC and the TCA cycle regulates mitochondrial bioenergetics

what does NADH and FADHs made in the mitochondria serve as?

-they serve as electron donors for the electron transport chain

supplements with B vitamins make claims that they increase energy output. Is this accurate?

-closer to something in between

-but as long as we have these vitamins, no matter how much we take, we will not get more energy, we get energy out of those fuels vitamins on there own can not provide energy but they can allow u to extract energy, but if you have enough of them it won't give you a boost beyond normal

**also if you don’t have these all these processes will not occur

what are the 3 regulatory enzymes of CAC?

1. citrate synthase (acetyl CoA + OAA to citrate)

2. isocitrate dehydrogenase (isocitrate to alpha-ketoglutarate)

3. alpha-ketoglutarate dehydrogenase complex (alpha-ketoglutarate to succinyl-CoA)

how is citrate synthase regulated?

-inhibited by high levels of citrate

how is alpha-ketoglutarate dehydrogenase complex regulated?

-inhibited by NADH

-stimulated by Ca2+

how is isocitrate dehydrogenase regulated?

-inhibited by NADH

-stimulated by ADP and Ca2+