Glycolysis & Metabolism

What is the relationship between [AMP] and [ADP] in the cell? (so therefore how AMP responds to ATP hydrolysis)

2ADP —→ ATP + AMP

[AMP] = [ADP]²/[ATP]

10 fold increase in ADP (Due to ATP hydrolysis e.g. muscle contraction) = 100fold increase in [AMP]

What influence does AMP have on glycolysis?

• Displaces ATP from 6PF1K, activating it

• Pushes forward

• F6P —> F16BP

• 6PF1K hydrolyses ATP in the process, releasing ADP that amplifies AMP in positive feedback

What is the bifunctional enzyme that modulates production of F26BP from F6P

6PF2K / F26BPase

What does F26BP modulate and what are the consequences?

• Negative at F16BPase

• Allosteric binding at 6PF1K makes active site have greater affinity for F6P, less affinity for ATP, activating

• Promotes glycolysis whilst repressing gluconeogenesis

What impact does glucagon have on the glycolytic/gluconeogenic balance of the liver (fully)?

• GPCRs are Gs and Gq

• Gs increase adenylyl cyclase activity

• Increase cAMP

• PKA activation (R and C dissociation)

• PKA phosphorylates bifunctional enzyme at 6PF2K, repressing kinase, activating phosphatase

• Decrease [F26BP]

• Decrease repression of F16BPase

• Coordinate response in activating gluconeogenesis, represses glycolysis

Draw a diagram showing the cycle between F6P and F16BP as well as the enzymes involved in this

https://ibb.co/prdvRDGy

What are the six main mechanisms of enzyme control?

• Substrate level control

• Cooperativity

• Allostery

• Covalent modification

• Compartmentation

• Gene expression

Simply, what three mechanisms of enzyme control does glucokinase show?

• Substrate level

• Cooperativity

• Compartmentation

How does glucokinase show substrate-level enzyme control?

• High Km for glucose so rate is dependent on [glucose]

• At higher concentrations, converts to high affinity form (coopertivity)

How does glucokinase show cooperative enzyme control?

• As [glucose] increases, slowly converts to high affinity form

How does glucokinase show compartmentation as enzyme control?

• Low blood glucose causes sequestering of GK in nucleus by GKRP, released when glucose levels raise

What two enzymes are good examples for allosteric regulation?

• F26BPase/6PF2K

• PK

Define feedforward stimulation

• metabolite produced early in a pathway activates an enzyme that catalyzes a reaction further down the pathway

What is PK allosterically activated / inhibited by?

• Activated by F16BP (saturating levels)

• Negative by ATP, alanine

How does activated PKA have its effect on glycolysis in the liver?

• Phos and inhibits PK-L, downregulating glycolysis

• Phos bifunctional enzyme, inhibiting kinase activity

• Decreases [F26BP]

• Less inhibition of F16BPase

• More gluconeogenesis

Phosphorylation of enzymes is an example of

• Covalent modification

How does PKA activation have a long-term effect on glycolysis / gluconeogenesis?

• Some activated PKA translocates to nucleus

• Phosphorylates TFs CREB (activates) and ChREBP (inactivates)

• Glucogenesis enzymes e.g. PEPCK, F16BPase, G6Pase expressed

• Glycolysis enzymes e.g. PK decreasd

• Longer term increase in gluconeogenesis

What is the Km of glucokinase and normal glucose levels?

• 10mM

• 5 mM

What is special about glucokinase?

• Concentration of its substrate under physiological conditions is able to act as signal for regulation of its rate due to high Km and cooperativity as it converts to high affinity forms

• Rate will change as glucose concentration fluctuates

What are the three irreversible reactions that need to be known?

• G —→ G6P

• F6P ——> F16BP

• PEP ——> Pyruvate

What enzymes are involved in the irreversible reactions between G ←—> G6P

• Glucokinase (in liver) hexokinase (in skeletal muscle)

• G6Pase

What enzymes are involved in the irreversible reactions between F6P ←—> F16BP

• F16BP

• 6PF1K

What enzymes are involved in the irreversible reactions between PEP ←—> pyruvate

• PK (pyruvate kinase)

• PEPCK

What does PEP / PEPCK stand for?

• Phosphoenolpyruvate

• carboxykinase

Draw a diagram showing the cycle between PEP and pyruvate as well as the enzymes involved in this

https://ibb.co/0pcXRQSp

What are the different ways of inactivating PK into PK-Pi?

• PKA phosphorylation (in response to glucagon activation)

• Ca2+-calmodulin dependent protein kinase can phosphorylate at both serine and threonine residues

What dephosphorylates PK-Pi back into active Pi?

• Protein phosphatase 2A when activated in downstream cascades from insulin:receptor binding

Hepatocytes have increased expression of glucagon receptors. What GPCRs are they and what does this do?

• Gs stimulates adenylyl cyclase, increase cAMP, activates PKA

• Gq activates PLCgamma, increased PIP2 hydrolysis, increase [Ca2+]

Describe how the Michaelis Menten graph of [glucose]against Vmax shows that glucokinase activity is both due to its Km and its cooperativity

• If was just due to high Km, would be a hyperbolic curve

• GK curve is actually sigmoidal

• Due to additive effect of slow interconversion into high affinity form as [glucose] increases (cooperativity)

What is the metabolic response to hypoxia?

• Induction of hypoxia-inducible transcription factor HIF-1

• Helix-loop-helix TF

• Increases expression of genes that encode glycolytic enzymes

p53’s influence on metabolism

• Inhibits glycolysis by activating TIGAR (F26BPase activity, decreases F16BPase inhibition)

• Diverts flux to pentose phosphate pathway, NADPH production and reduction of ROS - damage reduction

Tumour metabolism

• Express splice isoform of PK: PKM2 (usually embryonic)

• pTyr motifs produced by GF signaling, can bind, allosteric activator F16BP released and PKM2 inhibited

• Activated PKA can phosphorylate PKM2 at Y105, inactivating

• Accumulation of upstream glycolytic intermediates

• Diversion of glucose into lipid synthesis

Draw the graph of hexokinase vs glucokinase

https://ibb.co/m5b7shVq

Hexokinase is used in…

• Skeletal muscle (all tissue(

Glucokinase is used in…

• Liver, beta cells of pancreas

What is hexokinase inhibited by?

• Allosteric product inhibition (G6P)

Compare regulation of hexokinase vs glucokinase

• HK has allosteric product inhibition (G6P) and is not induced by insulin

• GK is induced by insulin, doesn’t experience product inhibition

• HK low Km so acts at low [gluc] and constant rate at physiological [], GK high Km so effect only seen when blood [] is high, rate and isoform affinity is [glucose] dependent

Simply, what are the three ways phosphorylation can affect proteins during metabolism?

• Activation / inactivation of enzymes

• Control of protein-protein interactions

• Longer term regulation of gene expression

Where does cAMP bind on PKA?

• 2x on each regulatory subunit

What enzymes is CREB a transcription factor for?

• PEPCK

• F16BPase

• G6Pasee

• Increase gluconeogenesis

What enzymes is ChREB a transcription factor for?

• PK

• Increase glycolysis

Simply, which enzymes can be activated by phosphorylation?

• Phosphorylase kinase

• Phosphorylase

• PK-L

Describe how adrenaline binding causes glycogenolysis in skeletal muscle

• Gs betaadrR

• Adenylyl cyclase increases cAMP

• PKA activation

• Phos phosphorylase kinase, activates

• Phos phosphorylase, activates

• Glycogen breakdown

Describe how phosphorylation can control protein-protein interactions

• PP1 dephosphorylalates phosphorylase kinase and phosphorylase to stop glycogen breakdown

• PP1 needs to be shuttled to glycogen by G protein

• PKA phosphorylates G protein, blocking interaction with PP1

• PKA phosphorylates inhibitor proteins, enabling interaction with PP1

What are the key small molecule regulators in the skeletal muscle and the liver, and why?

• AMP in muscle, F26BP liver

• Muscle is designed to respond to AMP produced in muscle contraction when ATP is low and has to be made by myokinase from 2ADP

• Not much AMP flux in liver as metabolic rate constant

• F26BP is modulated in response to hormone binding, which fluctuates more in liver

Explain differences between PK-M1/2 and PK-L

• Muscle vs liver isoforms

• PK-M1 lacks phosphorylation site for PKA, so Adr binding just increases glycogenolysis in muscle

• PK-M2 can be activated by PKA phosphorylation (Tyr) during foetal and tumour growth (incomplete oxidation of glucose is primary metabolism)

• PK-L is inactivated by PKA phosphorylation (S12), increasing gluconeogenesis in response to Adr or glucagon binding

Why is alanine an allosteric inhibitor of pyruvate kinase?

• Pyruvate turned into alanine or lactate when not directly exported to mitochondria

• High alanine indicative that lots of pyruvate is being made

• Negative feedback, homeostasis

Difference between liver and skeletal muscle bifunctional enzyme?

• Liver’s can be phosphorylated by PKA, decreasing F26BP levels in response to hormone binding

• SM cannot be phosphorylated by PKA

What 3 main enzymes are expressed differently between SM and liver?

• SM: hexokinase, non-phosphorylatable bifunc enzyme, PK-M (not phosphorylatable by PKA)

• Liver: glucokinase, phosphorylatable bifunc enzyme, PK-L (phosphorylatable by PKA)

Advantage of the difference in expression of HK/GK between skeletal muscle and liver

• Allows skeletal muscle glycolysis regardless of [glucose]

• Allows liver to respond to [glucose] to alter glycolysis according to fast or feeding states

Advantage of the difference in expression of the bifunctional enzyme between skeletal muscle and liver

• Allows liver to respond to PKA activation e.g. adr/glucagon to decrease glycolysis and coordinately increase gluconeogenesis to increase [glucose] in blood

• Skeletal muscle repsond to adr should be to increase glycolysis

Advantage of the difference in expression of pyruvate kinase between skeletal muscle and liver

• Want liver to increase gluconeogenesis so more glucose can be released into blood in response to Adr

• Want skeletal muscle to increase glycolysis in response to adr (fight or flight)

What are the four main roles of AMP in skeletal muscle?

• ‘Energy sensor’ due to concentration sensitivity in muscle contraction

• Activation of phosphorylase for more glycogen breakdown

• Allosteric inhibition of F16BPase to increase glycolysis flux

• Allosteric activation of 6PF1K by displacing ATP

• Activate AMPKK

What happens when AMP binds AMPKK?

• AMPKK phosphorylates AMPK

• Increase glucose transport