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1. BIOC 1301 S2 > Glycolysis > Flashcards

Glycolysis Flashcards

1
Q

What is metabolism?

A

The overall process by which living systems acquire and use energy to carry out their functions

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2
Q

What are the types of metabolism?

A

Catabolism - breakdown of nutrients to generate energy and provide raw materials

Anabolism - synthesis of biomolecules from simple building block

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3
Q

How is metabolism managed?

A

As the oxidation of glucose to CO2 is highly exothermic:
oxidative metabolism is done step-wise to release the energy in managable chunks
The energy is stored in high energy intermediates: ATP, Coenzyme A and NADH

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4
Q

What is glycolysis?

A

The lysis of glucose
Going from glucose to pyruvate
Generating ATP and NADH

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5
Q

Give an overview of Glycolysis

A

First stage of the oxidation of glucose to carbon dioxide
Enzymes of glycolysis are located in the cytosol
Converts glucose (C6) to two molecules of pyruvate (C3)
Generates a net total of two ATP and two NADH
Does not require oxygen (can occur in anaerobic conditions)

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6
Q

What are some features of ATP?

A

Made from ribose, adenine and 3 phosphate groups
Phosphoanhydride bonds - unstable between phosphate groups holds ATP together
These bonds have large free energy change when cleaved
The molecules need energy to hold it’s self together against the repulsion of itself
Resonance stabilisation of the phosphoanhydride is less than it’s hydrolysis products
Therefore ATP ‘wants’ to be driven to ADP

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7
Q

What system is also in place to provide ATP?

A

Phosphocreatine system
ATP + Creatine ↔ ADP + Phosphocreatine
Phosphocreatine acts as an ATP buffer within cells - a store of ATP

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8
Q

What is Step 1 of glycolysis?

A

Glucose is phosphorylated
Glucose + ATP ↔ Glucose-6-phosphate + ADP + H+

Uses hexokinase with cofactor Mg 2+

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9
Q

Step 1 glycolysis - describe hexokinase?

A

Transfers phosphoryl groups between ATP and substrate
A ubiquitous enzyme
Undergoes a large conformational change when glucose binds - bringing ATP and C6 of glucose together
Excludes water from the active site, avoid the thermodynamically favourable hydrolysis of ATP (facilitating the nucleophilic reaction)
Negatively regulated - as the product (G6P) binds to a regulatory site results in a conformational change to switch off catalysis (allosteric regulation)

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10
Q

Step 1 glycolysis - use of cofactor Mg 2+?

A

Without Mg 2+ ATP would act as a competitive inhibitor of hexokinase
Mg 2+ shields the negative charges of ATP’s phosphate oxygen atoms
This makes the phosphorus atom more accessible for nucleophilic attack by the C6-OH of glucose

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11
Q

What is the point of step 1 of glycolysis?

A

Adding a phosphate group to the sugar is now negatively charged which makes it harder to diffuse out of the membrane, therefore traps glucose in the cell for the rest of metabolism

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12
Q

What is step 2 of glycolysis?

A

Glucose-6-phosphate undergoes isomerisation to fructose-6-phosphate (F6P)

Uses phosphoglucose isomerase (PGI)
Aldose to a ketose structure

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13
Q

Step 2 glycolysis - how does phosphoglucose isomerase catalyse the reaction?

A
  1. The substrate binds
  2. Acid -catalysed ring opening
    ○ An enzymatic acid, likely the amino group of a conserved lysine residue, catalyses the ring opening
  3. Base catalysis
    ○ A base thought to be His imidazole group, abstracts the acidic proton from C2 to form cis-enediolate intermediate
  4. The proton is replaced on C1 in an overall proton transfer with a medium
  5. Base-catalysed ring closure
    ○ The ring closes to form the product, which is then released to yield free enzyme and completing the catalytic cycle
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14
Q

Step 2 glycolysis - what does phosphoglucose isomerase do generally?

A

As G6P and F6P have very similar structures

PGI has three stages:
ring opening
isomerisation
ring closure

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15
Q

What is step 3 of glycolysis?

A

Fructose-6-phosphate is phosphorylated
Fructose-6-phosphate + ATP ↔ Fructose-1,6-bisphosphate + ADP + H+

Uses phosphofructokinase (PFK) and cofactor Mg 2+ 
This is the Rate Determining step
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16
Q

Step 3 glycolysis - how does phosphofructokinase work?

A

The enzyme catalyses a nucleophilic attack by the C1-OH group of F6P on the P atom in the ATP Mg 2+ complex

17
Q

What is step 4 of gycolysis?

A

Fructose-1,6-bisphosphate is cleaved into Dihydroxyacetone phosphate (DHAP) and Glyceraldehyde-3-phosphate (GAP)

Uses aldolase (fructose bisphosphate aldolase)

18
Q

Step 4 glycolysis - how does aldolase work generally?

A

It catalyses aldol cleavage - retro aldol condensation
Enolate intermediate is resonance stabilised, due to the electron withdrawing character of the carbonyl oxygen atom
To get aldol cleavage between C3 and C4 requires a carbonyl at C2 and a hydroxyl at C4

19
Q

Step 4 glycolysis - what is the mechanism of aldolase?

A
  1. The substrate FBP binds to enzyme
  2. FBP carbonyl group reacts with the amino group of the active site lysine to form an iminium cation (protonated Schiff base)
  3. The C3-C4 bond is cleaved, forming an enamine intermediate and releasing GAP
  4. Protonation and tautomerization of the enamine yield the iminium cation from the Schiff base
  5. Hydrolysis of the iminium cation release DHAP and regenerates the free enzyme
20
Q

Facts about aldolase in different organisms?

A

In animals and plants - this reaction proceeds via a covalent intermediate (covalent catalysis)
In bacteria and fungi - type II aldolase in which the enolate intermediate is stabilised by a divalent metal ion (metal ion catalysis)

21
Q

What can take place after step 4 of glycolysis?

A

You can convert between the two products of DHAP and GAP

As they are ketose-aldose isomers they can undergo isomerisation using triosephosphate isomerase (TIM)

22
Q

Features of TIM?

A

Triose phosphate isomerase (TIM)
Catalytically perfect enzyme as the rate of the reaction is diffusion controlled - this is the slowest step
It has a TIM barrel structure (8 beta sheets surrounded by 8 alpha helices) - which is a very stable arrangement and a conserved 10-residue loop that closes over the active site to stabilise the intermediate and prevent breakdown

23
Q

What is the most common direction catalysed by TIM?

A

As GAP is constantly removed by downstream glycolytic reactions
This drives the DHAP -> GAP reaction forwards

24
Q

What is step 5 of glycolysis?

A

Glyceraldehyde-3-phosphate is turned into 1,3-bisphosphoglycerate (1,3-BPG)
Glyceraldehyde-3-phosphate + NAD+ + Pi ⇌ 1,3-bisphosphoglycerate + NADH + H+

Uses glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
This is an aldehyde oxidation, exergonic reaction, that drives the synthesis of the high energy acyl phosphate intermediate (1,3-BPG)

25
Q

Step 5 glycolysis - how does GAPDH work?

A

Glyceraldehyde-3-phosphate dehydrogenase has a cysteine residue in the active site

  1. Substrate binds
  2. Addition of thiol to active site, acting a nucleophile - forming a thiohemiacetal
  3. Dehydrogenation (oxidation) - forming a acyl thioester intermediate (contains more energy than a normal ester)
  4. Phosphate binding - phosphorolysis - to the enzyme-thioester-NADH complex
  5. Product is released and NADH/NAD+ exchange takes place to regenerate the enzyme
26
Q

What is step 6 of glycolysis?

A

1,3-bisphosphoglycerate is converted into 3-phosphoglycerate (3PG)
1,3-bisphosphoglycerate + ADP ⇌ 3-phosphoglycerate + ATP

Uses phosphoglycerate kinase (PGK) and a Mg 2+ cofactor
This is a coupled reaction with the ADP reaction being more exergonic therefore driving the overall reaction as exergonic, making the reaction feasible

27
Q

Step 6 glycolysis - PGK?

A

Phosphoglycerate kinase
generates the first ATP, following induced fit
Similar to hexokinase

28
Q

What is step 7 of glycolysis?

A

3-phosphoglycerate is converted to 2-phosphoglycerate (2GP)

Uses phosphoglycerate mutase (PGM)

29
Q

Step 7 glycolysis - PGM?

A

Phosphoglycerate mutase
Mutases move a functional group around within a molecule
Active enzyme has a phosphorylated histidine (His 8)
This phosphate is added to C2 of 3PG to make 2,3-bis-phosphoglycerate
The phosphate on C3 is then removed by the enzyme to regenerate phosphohistidine and yield 2PG

30
Q

What is step 8 of glycolysis?

A

2-phosphoglycerate is converted into phosphoenolpyruvate (PEP)
2-phosphoglycerate ⇌ phosphoenolpyruvate + H2O

Uses enolase
PEP is a high energy compound

31
Q

What is step 9 of glycolysis?

A

Phosphoenolpyruvate is converted into Pyruvate
Phosphoenolpyruvate + ADP + H+ ⇌ Pyruvate + ATP

Uses pyruvate kinase (PK)

32
Q

Step 9 glycolysis - describe pyruvate kinase generally?

A

Eukaryotic PK requires potassium
Bacterial PK has a conserved lysine thought to carry out the same function
The PK has two stages: hydrolysis and tautomerization
Highly exothermic and able to drive ATP synthesis

33
Q

Step 9 glycolysis - what does pyruvate kinase do exactly?

A

A beta-phosphoryl oxygen of ADP nucelophilically attacks the PEP phosphorus atom, therefore displacing enolpyruvate and forming ATP
Enolpyruvate tautomerizes to pyruvate

34
Q

What are the overall products of glycolysis?

A

2 ATP
2 NADH
2 Pyruvate

35
Q

What are the main regulatory points of glycolysis?

A

Phosphofructokinase

with also hexokinase and pyruvate kinase

36
Q

Describe phosphofructokinase?

A

PFK is a tetramer with two conformational states R and T
R state - substrate binding (Arg162 binds to F6P)
T state - repels the substrate: Arg162 is replaced by Glu161 (two negative charges repel)
It also has two binding sites for ATP (as ATP is botha substrate and an allosteric inhibitor)

37
Q

How is phosphofructokinase controlled?

A

ATP binds in the T state (inactive) and F6P binds in the R state (active)
At high concentrations of ATP, this shifts the equilibrium to favour the T state - decreasing PFKs affinity for F6P

Activators (AMP, ADP) bind to the R state

38
Q

Overview of metabolic control?

A

Low metabolic demand - ATP is high, PFK inhibited, flux through glycolysis is low
High metabolic demand - ATP is low, PFK active, Flux through glycolysis is high
ATP concentration varies by 10% between rest and vigorous activity

AMP is a better indicator of low/high levels for metabolic demand
10% change in ATP = 100% change in ADP = >400% change in AMP
Adenylate kinase catalyses the reaction of 2ADP ⇌ ATP + AMP

1. BIOC 1301 S2 (23 decks)

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