7/26/2021

Make sure you understand

how enzymes and pathways
contribute to metabolism

Excellent example of how

enzymes work

https://www.youtube.com/watc
h?v=yk14dOOvwMk

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A refresher on REDOX and

metabolism

Oxidation reactions extract

energy from bonds!

• Note: energy from C-C bonds = C-H

bonds, but C-Cs are not oxidised
directly?

• Molecules must be rearranged to C-H

or C-OH for oxidation!

• The electrons from these bonds

ultimately flow to oxygen (or other compounds in
odd critters)

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Leaving hydrogens take

electrons

malate oxaloacetate

REDOX reactions use

DEHYDROGENASES!
Note this is a silly name as they can usually work in
the reverse direction and hydrogenate as well

Another explanation?

Energy release must be controlled

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1/
H2 + 1/2 O2 2H 2 O2
(from food via NADH)
Controlled
release of
2 H+ + 2 e– energy for
synthesis of
ATP

Explosive
release of
heat and light
energy

1/
2 O2

(a) Uncontrolled reaction (b) Cellular respiration

Cellular respiration is controlled energy captured in ATP

7

So what is glycolysis?
• Sugar splitting (glyco-lysis) requires a pathway
• Glycolysis is universal, it is ancient
• Other sugars can be used, … but we’ll stick
with glucose
• Glucose, - 6 carbon (6C) molecule is trapped
using phosphate, rearranged and split into 2 x
3C molecules
• 2 ATP are first invested, and 4 ATP harvested
• Net 2 ATP are made
• Electrons are also striped (there is an oxidation)

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The Stages of Cellular

Respiration: A Preview

• Cellular respiration has three stages:

– Glycolysis (breaks down glucose into
two pyruvate molecules)
– The Citric Acid Cycle (completes the
breakdown of glucose)
– Oxidative phosphorylation (accounts
for most of the ATP synthesis)

Fig. 9-6-3
Now we focus on this bit

Electrons Electrons carried

carried via NADH and
via NADH FADH2

1 2 Oxidative
Glycolysis Citric phosphorylation:
acid electron transport
Glucose Pyruvate cycle and

3 chemiosmosis

Mitochondrion
Cytosol

ATP ATP ATP

Substrate-level Substrate-level Oxidative

phosphorylation phosphorylation phosphorylation

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1
Glycolysis
2

3 10 enzymes
4, 5 produces 2 ATP net
only one oxidation
reaction rearranges &
prepares
6 for respiration
7

8 You don’t need to know

structures
9 but they may help!

10 From: Biochemistry, 5th Edition

Berg, Tymoczico and Stryer

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Schematic representation of facilitated transport of

glucose through the cell membrane.

Na+ is also required for glucose uptake

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Conversion of
glucose to
glucose- 6-
phosphate

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The KM and affinity?

• A high KM = a low affinity

• If glucokinase has a higher KM than
hexokinase, does this mean GK binds
glucose better than HK at a low
concentration!

• Yes or No?

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Glucose

ATP
1
Hexokinase

ADP

Glucose
Glucose-6-phosphate

ATP
1
Hexokinase

ADP
Glucose now trapped
in the cell
Phosphate is charged
and glucose is no
longer recognised by
GLUTs
Glucose-6-Phosphate
Glucose-6-phosphate is now tagged for other
things

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Glucose

ATP
1
Hexokinase

ADP

Glucose-6-phosphate
Glucose-6-phosphate

2
Phosphoglucoisomerase

2
Fructose-6-phosphate
Phosphogluco-
isomerase

rearrangement

Fructose-6-phosphate
Why the rearrangement?...

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Glucose

ATP
1
Hexokinase

ADP

Fructose-6-phosphate …In preparation

Glucose-6-phosphate

2
for the next step
Phosphoglucoisomerase

ATP Commitment &

3 investment
Fructose-6-phosphate
Phosphofructo-
ATP
kinase
3
Phosphofructokinase ADP
ADP

Fructose-
1, 6-bisphosphate

Fructose-
1, 6-bisphosphate

Glucose is now fully committed to catabolism (breakdown)

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Not quite symmetrical so another

Glucose
rearrangement occurs
ATP
1
Hexokinase

ADP

Glucose-6-phosphate

2
Phosphoglucoisomerase
Fructose-
1, 6-bisphosphate
4
Fructose-6-phosphate
Aldolase
ATP
3

Rearrangement
Phosphofructokinase

ADP

5
Triose phosphate
Isomerase
Fructose-
1, 6-bisphosphate

4
Aldolase

5
Isomerase

Dihydroxyacetone Glyceraldehyde-
phosphate 3-phosphate
Dihydroxyacetone Glyceraldehyde-
phosphate 3-phosphate

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2 NAD+ 6
GAPDH

2 NADH 2 Pi
+ 2 H+

2 1, 3-Bisphosphoglycerate

Glyceraldehyde-
3-phosphate
6
2 NAD+
Energy Harvest Glyceraldehyde-3-
phosphate
begins…but… dehydrogenase There is an
2 NADH 2 Pi injection of Pi
this uses no ATP
+ H+
Redox reaction
An oxidation
(the only one in
glycolysis) … the energy
The cofactor harvest here can
NADH is used 2 1, 3-Bisphosphoglycerate not be used in us
without
oxygen…
LATER

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REDOX reactions use dehydrogenases

(de-hydrogen-ase)

So NAD+ acts as an
electron shuttle

In glycolysis NADH + H+ is
formed by glyceraldehyde-3-
phosphate dehydrogenase
(GAPDH)

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2 NAD+ 6
Triose phosphate
dehydrogenase
2 NADH 2 Pi
+ 2 H+

2 1, 3-Bisphosphoglycerate
2 ADP

7
Phosphoglycerokinase
2 ATP

2 1, 3-Bisphosphoglycerate
2 ADP
2 3-Phosphoglycerate
7 The first return
Phosphoglycero- of ATP x 2 (for
2 ATP kinase
each triose)

A A
2 3-Phosphoglycerate

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2 NAD+ 6
Triose phosphate
dehydrogenase
2 NADH 2 Pi
+ 2 H+

2 1, 3-Bisphosphoglycerate
2 ADP
7 Phosphoglycerokinase

2 ATP

3-Phosphoglycerate
2 3-Phosphoglycerate

8
2
Phosphoglyceromutase
The phosphate is moved
8
Phosphoglycero-
2 2-Phosphoglycerate mutase

2 2-Phosphoglycerate

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2 NAD+ 6
Triose phosphate
dehydrogenase

2 NADH 2 Pi
+ 2 H+

2 1, 3-Bisphosphoglycerate
2 ADP
7 Phosphoglycerokinase

2 ATP

2 3-Phosphoglycerate 2 2-Phosphoglycerate
8
Phosphoglyceromutase

9
Enolase
2 H2O
2 2-Phosphoglycerate

9
Enolase
2 H2O

2 Phosphoenolpyruvate

Dehydration (H2O)

2 Phosphoenolpyruvate

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2 NAD+ 6
Triose phosphate
dehydrogenase

2 NADH 2 Pi

+ 2 H+

2 1, 3-Bisphosphoglycerate
2 ADP
7 Phosphoglycerokinase

2 ATP

2 Phosphoenolpyruvate
2 ADP
2 3-Phosphoglycerate

8
Phosphoglyceromutase 10
Pyruvate
2 ATP kinase
2 2-Phosphoglycerate

9
Enolase
2 H2O

A A
2 Phosphoenolpyruvate
2 ADP
10
Pyruvate kinase
2 ATP

2 Pyruvate

2 Pyruvate

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Stages of
glycolysis

Two phases,
1st) Investment, -2 ATP

2nd) Payoff harvest, +4 ATP

4ATP – 2ATP =
2 ATP net!
From: Biochemistry, 5th Edition,
Berg, Tymoczico and Stryer

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The energy
input and Energy investment phase

output of Glucose
glycolysis
2 ADP + 2 P 2 ATP used
An overview

Energy payoff phase

4 ADP + 4 P 4 ATP formed

2 NAD+ + 4 e– + 4 H+ 2 NADH + 2 H+

2 Pyruvate + 2 H2O

Net
Glucose 2 Pyruvate + 2 H2O
4 ATP formed – 2 ATP used 2 ATP
2 NAD+ + 4 e– + 4 H+ 2 NADH + 2 H+

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Complicated yes!
But life just isn't easy

Just remember
10 enzymes
glucose is trapping
NADH NADH investment (2 ATP) & commitment
splitting
Pi addition of Pi with oxidation
Pi
A A harvest 4 ATP but invested 2 ATP
4–2=2
Net gain = 2 ATP &
A A 2NADH

27

Substrate level
phosphorylation

NADH NADH

Pi Pi
A A
Phosphoglycerate kinase
Note these kinases were
inappropriately named
A A
Pyruvate kinase

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The enzymes
Hexokinase or
3 Glucokinase (1st)

Phosphofructokinase
(3rd)

Pyruvate Kinase
(10th)

Highly regulated and

irreversible in vivo

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PFK
Glucose is now
committed
Highly regulated point in
pathway
Another ATP invested

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Phosphofructokinase (PFK)
Most important regulator

PFK is regulated by:

• High ATP (why make more and waste
glucose?)
• AMP activates PFK (i.e. not very much
ATP, make more!)
• Citrate inhibits PFK from the CAC (later)
• Acidification inhibits PFK (save fuel, demands
too high on metabolism?)

PFK is the first committed step, there is no

going back, PFK is not reversible
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The two fates of glucose (with

O2 without O2)

& Glycolysis can be anaerobic

i.e. Without O2
Glycolysis can feed into aerobic,
(this is much less efficient ~15 x
Pathways
less ATP, but usually fast ~100 x
(i.e. With O2)
faster!)

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• Generally electrons in NADH

ultimately are used in aerobic
respiration…
• but what if there is no O2?
• Remember there was likely
limited O2 when life evolved
• There are problems if there
is no oxygen, there is only
so much NAD!

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NAD is regenerated by LDH

2
Production of
lactate
during
anaerobic
glycolysis
From: Exercise Physiology, 5th Edition, McArdle, Katch and Katch

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Glucose
NAD+ is reformed glycolysis
continues (well.. longer till
lactate accumulates)

GAPDH

The +enzyme involved is Lactate +

NAD NADH
Dehydrogenase
Pyruvate
(LDH)

Lactate Lactate LDH

Lactate Lactate
Lactate Lactate
Lactate Lactate Lactate
Lactate Lactate Lactate
Lactate Lactate Lactate
Lactate Lactate Lactate
Lactate Lactate Lactate

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Blood lactate as an energy source: the Cori

cycle

From: Biochemistry, 5th Edition, Berg, Tymoczko and Strye

36

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Alternatives to lactate

CO2

From: Biochemistry, 5th Edition, Berg, Tymoczico and

Stryer

Yeast ferment glucose to

ethanol

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Energy yield of glycolysis

• ATP releases 30-70 kJ/mol
• At 2 mole ATP  60~140 kJ
• Glucose contains 2870 kJ/mole
• 60 or 140 / 2870 x 100
• Only 2-4.5% efficiency!
Energy yield of aerobic glucose oxidation

• 31-73% efficiency!
• Much, much better!

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Things to do
If the following REDOX reaction occurred,
which compound would be reduced and
which is oxidised?

C3H5O6P + NAD+ + Pi  C3H4O10P2 + NADH

•
•
•
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In glycolysis phosphofructokinase (PFK) is

a highly regulated step and is allosterically
regulated by ATP.

What do you think very high concentrations of

ATP will do to the activity of PFK?

Be careful in your interpretation, as ATP is

also a substrate of PFK (HMMMM???).

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In glycolysis, aldolase splits fructose

1,6-phosphate to form
dihydroxyacetone phosphate
(DHAP) to glyceraldehyde 3-
phosphate (GA3P).

Triose phosphate isomerase (TPI)

then converts DHAP to GA3P. If
there is a side reaction that
steals/removes dihydroxyacetone
phosphate, what could happen?

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