Focus on Glycolysis

Question 1

What are the 3 stages of catabolism in the presence of oxygen?

Answer 1

1- Acetyl-CoA production - glucose, fatty acids and amino acids all converge to make simple 2C Acetyl-CoA
2-Acetyl-CoA oxidation - citric acid cycle
3-Electron transfer and oxidative phosphorylation

Question 2

Whta happens carbs, fats and proteins when digested and absorbed by chemotrophs?

Answer 2

-used immediately for energy via catabolic pathways
-or synthesised into glycogen (glucose store) - fuel
-or triacyclglycerols (fat store) for storgae and later catabolism - fuel

Question 3

What is the major fuel in most organisms that plays a central role in metabolism and why?

Answer 3

D-Glucose (alpha form)
-completre oxidation of one d-glucose to CO2 and H2O after 3 stages of catabolism
-gives mega-energy - -2840kJ/mol

Question 4

4 major pathways of glucose utilisation?

Answer 4

1. storage -> glycogen, starch, sucrose
2. oxidation via glycolysis -> pyruvate
3. oxidation via pentose phosphate pathway
   -> ribose 5-phosphate
4. synthesis of structural polymers -> extracellular matrix, cell wall polysaccharides

Question 5

Points on glycolysis?

Answer 5

-1st metabolic pathway elucidated in yeast and muscle cells
-near universal pathway
-responsible for largest flux of C in cells
-role of ATP and phosphorylated compounds in cell biology discovered by research which discovered glycolysis

Question 6

What happens in the Preparatory Phase in glycolysis? - 5 steps

Answer 6

1. Glucose is phosphorylated by hexokinase to form glucose 6-phosphate
2. Glucose 6-phosphate undergoes isomerisation by phosphohexose isomerase to form Fructose 6-phosphate
3. Fructose 6-phosphate is phosphorylated by phosphofructokinase-1 to form Fructose 1,6 bisphosphate
4. Fructose 1,6 bisphosphate is cleaved by aldose into Glyceraldehyde 3 phosphate and Dihydroxyacetone phosphate
5. Glyceraldehyde 3-phosphate and Dihydroxyacteone phosphate are interconversed by triose phosphate isomerase to form Glyceraldehyde 3-phosphate

Question 7

What happens in the Pay Off Phase in Glycolysis? -5 steps

Answer 7

6. Glyceraldehyde 3- phosphate is oxidised and phosphorylated by Glyceraldehyde 3-phosphate dehydrogenase to form 1, 3-biphosphoglycerate
7. 1, 3-biphosphoglycerate is involved in a substrate phosphorylation by phospho-glycerate kinase to release ATP and form 3 phosphoglycerate. ATP PRODUCTION
8. 3 phosphoglycerate becomes 2 phosphoglycerate due to phosphoglycerate mutase
9. A water molecule is removed from 2 phosphoglycerate by enolase to form phosphoenol pyruvate
10. Phosphoenolpyruvate undergoes substrate level phosphorylation by pyruvate kinase to produce ATP and pyruvate- ATP PRODUCTION

Question 8

What happens in step 1 of glycolysis and what does this involve?

Answer 8

Hexokinase
Glucose is phosphorylated on position 6 - ATP donates the phosphate
-Priming irreversible reaction - ATP is consumed in order to energise the system - destabilises glucose for breakdown by phosphorylating it
-Free energy becomes large and negative as ATP adds its terminal phosphate onto glucose

Question 9

What are kinases?

Answer 9

A large family of enzymes that add phosphoryl groups to substrates - substrate level phosphorylation

Question 10

What does Hexokinase require and why?

Answer 10

Requires Mg2+ as it shields the terminal phosphate of ATP from hydrolysis by H20 to enable nucleophilic attack by OH on C6 of glucose

Question 11

Structure of hexokinase?

Answer 11

-Dimer and has 4 isoforms made from different genes
-Regulatory enzyme of glycolysis
-Has 2 active sites - one is regulatory site and is is phosphorylating
-Has a U-shaped clamp like structure - conformational change with clamp closing induced by binding of D-glucose
-The phosphorylation of glucose by HK traps glucose inside cells for metabolism

Question 12

What happens in step 2 - Phosphohexose isomerase?

Answer 12

Glucose 6-phosphate isomerises to Fructose 6-phosphate
-near-equilibrium rxn - reversible
-small chnage in standard free energy
-reversible isomerisation of an aldose to a ketose
-shuffles atoms around to move carbonyl from C1 to C2 so next reaction can happen

Question 13

What happens in step 3 - Phosphofructokinase-1 (PFK1)

Answer 13

Fructose 6-phosphate is phosphorylated tp Fructose 1,6 bisphosphate ATP dontaes the phosphate
-Primining irreversible rxn
-ATP consumed here again as in step 1 - neg free energy
-committed step for glycolysis - rate limiting step

Question 14

What is PFK-1?

Answer 14

Majore regulatory enzyme of glycolysis

Question 15

What happens in step 4 - enyzme aldolase?

Answer 15

Hexose is lysed (split) to form 2 trioses - Dihydroxyacetone phosphate and Glyceraldehyde 3-phosphate
-pos change in free energy - reversible rxn
-near equilibrium rxn
-C1 to C3 of Fructose 1, 6 bisphosphate makes DHAP
-C4 to C6 of F1,6-BP makes G3-P
-Lysine residue important for lysis in enzyme

Question 16

What happens in step 5 - Triose Phosphate isomerase (TPI)

Answer 16

Isomerisation of DHAP tp G3P
-Near equilibrium rxn - interchangeable forms
-TPI pulls H off of one carbon and replaces it onto another carbon
-called a perfect enzyme
-changes molecule into useable form

Question 17

What happens in step 6 - Glyceraldehyde 3-phosphate dehydrogenase?

Answer 17

2 glyceraldehyde 3 phosphate oxidised and phosphorylated to 1,3 bisphosphoglycerate
-oxidation transfers electrons from 2 x G3P to 2NAD to form 2NADH - energy
-prepares for energy production as v high energy of hydrolysis of 1,3 BPG

Question 18

What happens in step 7 - Phosphoglycerate kinase?

Answer 18

-2 x 1,3 phosphoglycerate is dephosphorylated at position 1 to yield 2 x 3-phosphoglycerate and 2 ATP
-Substrate level phosphorylation

Question 19

What role does the induced fit motion in phosphoglycerate kinase play?

Answer 19

-Same as hexokinase
-Closes around rxn, protecting it from interfering with water molecules so ATP is not hydrolysed immediately
-2 lobes - upper lobe binds ADP and lower pocket binds 1,3BPG - hinges closes to perform transfer of phosphate

Question 20

What happens in step 8 - Phosphoglucomutase?

Answer 20

2 x 3 phosphoglycerate isomerises to 2 x 2 phosphoglycerate
-the phosphate group transfers onto enzyme active site before it moves to position 2
-in presence of magnesium
-creates a more amenable susbtrate for rxn 9

Question 21

What happens in step 9 - Enolase?

Answer 21

-2 x 3 phosphoglycerate dehydrated to yield 2 x phosphoenolpyruvate and 2 x H2O
-this dehydration places the phosphate in an uncomfortable position making it easy to remove to form ATP in next rxn - the new double bond is in an awkward place
-Phosphoenolpyruvate is a high energy molecule

Question 22

What happens in step 10 - Pyruvate kinase?

Answer 22

-2 x phosphoenolpyruvate is dephosphorylated to yield 2 x pyruvate and 2 ATP
-2nd substrate level phosphorylation
-regulatory enzyme

Question 23

Structure of pyruvate kinase?

Answer 23

-An allosteric enzyme
-4 flexible subunits arranged in a diamound shape
-Senses levels of energy metabolites and turns on/off according to energy needs
-these allosteric modulators chnage shape of enzyme active <-> inactive

Question 24

What is the total energy gained from glycolysis and what is this pathway?

Answer 24

2ATP and 2NADH
Exergonic pathway
(2 ATP invested and 4 produced)

Question 25

Why is this pathway unusual?

Answer 25

As things not usually broken down in the cytosol

Question 26

Why is the foirmation of glucose -6 phosphate not a rate limiting step (first step)?

Answer 26

As can be used in the cell anways in that form -second phosphorylation is rate limiting step- the PFK-1

Question 27

What is the 3 possible fates of pyruvate?

Answer 27

In presence of oxygen - becomes 2 Acetyl CoA and enters citric acid cycle Absence of oxygen - ethanol and CO2 (in yeast) or Lactate (in muscles)

Question 28

Fate of Pyruvate in presence of oxygen?

Answer 28

1. 2 pyruvate decarboxylated to 2 acetyl-CoA (2C) and 2CO2 2. 2 x acetyl-CoA enters citric acid cycle to form citrate oxidised with electron transfer to NAD and FAD - committed step 3. NADH and FADH2 transfer electrons to respiratory chain in to create ATP energy -All happens in mitochondria

Question 29

How muc total energy is porduced from oxidation of 1 glucose aerobicallly with the 3 stages?

Answer 29

2850kJ energy

Question 30

In anerobic conditions what enzymes produce lactate and ethanol from pyruvate?

Answer 30

Pyruvate - lactate dehydrogenase - lactate Pyruvate - pyruvate decarboxylase - acetaldehyde - alcohol dehydrogenase - ethanol

Question 31

Why cant vertebrates convert pyruvate to ethanol?

Answer 31

Pyruvate decarboxylase is absent in vertebrates and lactic acid bacteria

Question 31

What essential regeneration takes place in anaerobic conditions?

Answer 31

Regeneration of NAD+ which keeps stepp 6 glycolysis supplied with NAD+ allows continuous glycolysis to occur in absence of O2

Question 32

When does lactate production happen?

Answer 32

1. Wwide range of organisms in anaerobic niches produce lactate from glucose for energy fermentation of sugars to lactate by m/o used in cheese yogurt production 2. erythrocytes, retina, brain cells produce lactate even even O2 present - responsible for atleats 10% overall glucose breakdown 3. Very active skeletal muscle uses anaerobic pyruvate - lactate conversion when O2 cant get to muscle fast enough - anaerobic respiration 4. Cancer cells and immune cells can have very active anaerobic glucose catabolism even in presence of O2 (Warburg effect) for fast proliferation

Question 33

Whta happens when anaerobic respiration takes place in muscles?

Answer 33

-O2 cant get to muscle fast enough -Muscle switches to anaerobic glycolysis -Breaks down muscle glycogen stores -> ´glucose -> pyruvate -> lactate for energy -Lactate produced blood -> liver - Lactate synthesises glucose in liver -glucose goes from liver -> blood -> muscle replenishes glycogen

Question 34

What glycolytic enzymes can occur in cancer cells?

Answer 34

-Increased levels of glycolytic enzymes -Diff isoforms of glycolytic enzymes can occur including Pyruvate kinase PKM2 rther than PKM1 -PKM2 has reduced catalytic activity compared to PKM1 - allows cancer cells to keep using anaerobic glycolysis for energy and build up glycolytic intermediates that feed into synthetic pathways promoting proliferation