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Glycolysis, gluconeogenesis and the TCA cycle

Prof. Alison Baker

BLGY1115 L9

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TCA cycle

Glucose

Pyruvate

Acetyl CoA

Fatty acids

citrateAcetyl CoA

GlycolysisGluconeogenesis

Fatty acid synthesis

Fatty acid

degradation

NADH

FADH2

ATP

Overview of Metabolism

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Glycolysis-Takes place in the cytoplasm

Note –

which reactions require ATP

which synthesise ATP

Which synthesise NADH

many reactions are reversible

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Substrate level phosphorylation

A phosphate group is transferred from one compound to another withoutthe involvement of electron transfer reactions as occur when ATP is synthesised in mitochondria as we shall see later.

All that is required is that the standard free energy of hydrolysis Go’of the compound donating the phosphate group is larger and more negative

than the Go’ of hydrolysis of the phosphate containing compound that is formed.

1,3 bisphosphoglycerate → 3-phosphoglycerate Go’ = -49.3 kJ mol-1

ADP + Pi =H+→ ATP + H2O Go’ = + 30.5 kJ mol-1

The enzyme phosphoglycerate kinase couples these reactions allowing the large negative G of the first reaction to be used to drive the energetically unfavourable second reaction.

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Figure 2-72a Molecular Biology of the Cell (© Garland Science 2008)

Phosphoglyceratekinase

Glyceraldehyde 3 Phosphate dehydrogenase

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Figure 2-73 Molecular Biology of the Cell (© Garland Science 2008)

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Glycolysis net reaction

Glucose +2 ADP + 2Pi + 2NAD+ →2 pyruvate + 2ATP+ 2NADH

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Quick self test

find the WRONG answer

1. Glycolysis takes place in the cytoplasm

2. Uses 2 ATP per glucose at the beginning of

the pathway

3. Splits a 6 C sugar phosphate to two 3C sugar

phosphates

4. Yields 2 molecules of NADH per glucose

5. Yields 2 molecules of ATP by substrate level

phosphorylation

6. Overall yield is 2 ATP per glucose

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Glycolysis net reaction

Glucose +2 ADP + 2Pi + 2NAD+ →2 pyruvate + 2ATP+ 2NADH

Q. What happens to the pyruvate and NADH?

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Under anaerobic conditions NAD+ is regenerated

2 pyruvate 2 lactate

2NADH 2NAD+

e.g in muscle

2 pyruvate 2 ethanol + 2 CO2

2NADH 2NAD+

e.g alcoholic fermentation in yeast

More on fermentation from Prof Denecke later

This is essential for anaerobic glycolysis to be sustained

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Cori cycle

Glucose

Pyruvate

Lactate

2ATP

2 NAD+

Muscle

Anaerobic glycolysis

blood

Glucose

Pyruvate

Lactate

4ATP

NADH

2 NADH

Liver

Gluconeogenesis

2 GTP

Lactate produced by anaerobic glycolysis in the sprinter’s musclesis transported to the liver and reconverted to glucose.

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Gluconeogenesis is not simply the reverse of glycolysis

ADP + Pi + PEP → pyruvate + ATP Pyruvate kinase (glycolysis)

Pyruvate + ATP + CO2 → oxaloacetate + ADP + Pipyruvate carboxylase (gluconeogenesis)

Oxaloacetate + GTP → PEP + GDP + CO2PEP carboxykinase (gluconeogenesis)

The pyruvate kinase reaction is essentially irreversible (Go’ -62.8 kJ mol-1) so 2

additional reactions using high energy phosphate compounds are required to bypass

this step.

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Gluconeogenesis proceeds by the reverse ofGlycolysis as far as fructose 1,6 bisphosphate

Two different enzymes,fructose 1,6 bisphosphatase andglucose 6 phosphatase are required to reverse the twoATP requiring steps of glycolysis catalysed by Hexokinase and phosphofructokinase

These enzymes are heavily regulated to avoid glycolysisand gluconeogenesis both being active at the same time

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Gluconeogenesis

Note –

use of 2 high energy phosphate bonds to bypass pyruvate kinase

distinct enzymes to reverse the two ATP requiring steps of glycolysis

oxaloacetate

ATP + CO2ADP + Pi

GTP

GDP + Pi + CO2

Glucose-6 phoaphatase

Fructose 1,6bisphosphatase

Pyruvate carboxylase

PEP carboxykinase

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Quick self test 2

find the WRONG answer

1. When there is insufficient oxygen pyruvate is converted to lactate.

2. Lactate formation allows oxidation of NADH so that glycolysis can continue

3. Lactate can be converted back to glucose in the liver

4. The pathway that does this is called gluconeogenesis

5. Gluconeogenesis is the exact reverse of glycolysis

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Glycolysis net reaction

Glucose +2 ADP + 2Pi + 2NAD+ →2 pyruvate + 2ATP+ 2NADH

Q. What happens to the pyruvate and NADH?

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Under aerobic conditions pyruvate and NADH from glycolysis are oxidised in mitochondria

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Site of TCA cycle

Site of electron transfer reactions and ATP

Synthesis. Highly impermeable.

Permeable to molecules of up to 1000 Da

The impermeability of the inner membrane is essential for ATP synthesis, but means

that transporter proteins are required to move metabolites in and out of mitochondria

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The inner membrane contains specific transport proteins forimportant metabolites

We shall return to how transport is energised in the next lecture.

20The malate-aspartate shuttle for transporting reducing equivalents

Malate dehydrogenase

Aspartate amino transferase

Aspartate amino transferase

Malate dehydrogenase

NAD+ and NADH do not have a transport protein butneed to cross the inner membrane

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Quick self test 3

The mitochondrial inner membrane is highly

impermeable and specific transporters are

required to move metabolites between the

matrix and the cytosol.

True or False?

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Pyruvate is transported into the mitochondrial matrix and converted to Acetyl CoA by Pyruvate Dehydogenase

This step is an important site of regulation of the TCA cycle (see later)

Pyruvate + NAD+ + CoA→ acetyl CoA + CO2 + NADH

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Overview of the TCA cycle

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The TCAcycle

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Can you work out what the net reaction of the cycle is?

Acetyl CoA + 3NAD+ +FAD + GDP + Pi +2H2O →CoASH +3(NADH +H+) +FADH2 +GTP +2CO2

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The TCA cycle is a source of biosynthetic precursors

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‘Topping up’ the cycle

If intermediates that are removed for biosynthesis are not replenished the cycle

will stop

Note that all carbon that enters as the acetyl group is oxidised to CO2, so

Simply putting in more acetyl CoA is no help

Pyruvate carboxylase provides a means of replenishing oxaloacetate

Pyruvate + CO2+ATP+H2O → Oxaolacetate + ADP +Pi + 2H+

We have met this reaction before in gluconeogenesis

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Control of the cycle

Pyruvate dehydrogenase

a-ketoglutarate

dehydrogenase

Succinyl CoA

synthetase

The products of the cycle,

ATP and NADH

inhibit it by activating

a protein kinase that

phosphorylates and inhibits

PDH

There is also regulation

of PDH activity by

hormones, e.g.

Insulin stimulates

PDH dephosphorylation

and therefore activation

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Quick self test 4

find the INCORRECT answer

1. The TCA cycle completely oxidises acetyl

groups supplied as acetyl CoA

2. The products of the TCA cycle are CO2, GTP,

NADH and FADH2 and CoA

3. Intermediates can be removed from the cycle

for biosynthetic reactions.

4. Pyruvate dehydrogenase converts pyruvate to

oxaloacetate allowing the intermediates of the

cycle to be topped up.

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Reading

• You will find the material covered in these 3 lectures in any good biochemistry text book.

• Some examples from the libraryEssential biochemistry / Charlotte W. Pratt, Kathleen Cornely.

• ed. Hoboken, N.J. : Wiley ; Chichester : John Wiley [distributor], c2011. c2011

• Biochemistry / Mary K. Campbell, Shawn O. Farrell.

•6th ed. : international student ed.

Pacific Grove, Calif. : Brooks/Cole ; London : Thomson Learning [distributor], c2009. c2009

• Principles of biochemistry / Donald Voet, Judith G. Voet, Charlotte W. Pratt.

•3rd ed., International student version.

Hoboken, N.J. : Wiley, c2008. c2008