Metabolism: Carbohydrate Metabolism: Inge Holsbeeks

Metabolism:
Carbohydrate
metabolism
Inge Holsbeeks
2nd stage bachelor industrial sciences

2019-20
Carbohydrate metabolism
• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Uptake of glucose
• Fermentation
• Pyruvate dehydrogenase
• Citric acid cycle
• Eletron transport chain
• ATP-synthase - Oxidative phosphorylation
2 5. Metabolism: Carbohydrate metabolism

Glucose is an important fuel for
most organisms

Glycolysis and connection to other
pathways
• an energy-conversion
pathway in many
organisms

Glycolysis and connection to other
pathways

Glycolysis
• 3 stages
• 10 steps
The glycolytic pathway can be divided into three stages: (1)

glucose is trapped and destabilized; (2) two interconvertible
three-carbon molecules are generated by cleavage of six-
carbon fructose; and (3) ATP is generated.

ATP- ATP-
hydrolysis hydrolysis
ATP-
synthesis
ATP ADP
ATP- fermentation
synthesis
reversible reactions + irreversible reactions → glycolysis is

irreversible
+ phosphorylation
ATP ADP NADH+H+ NAD+
fermentation
Zie fermentatie en ETC: NADH + H+  NAD+

Remark
• Interactive metabolic pathways

• Some enzyme categorisations are wrong
• E.g. hexokinase is not a transaminase but
transferase (of –P)

Step 1: glucose  glucose-6-P
Without enzyme
The phosphate groups in ATP are

shielded from nucleophilic attack
With enzyme – hexokinase - transferase

Step 2: Glu-6-P ↔ fruc-6-P
Glucose-6-P
Fructose-6-P
Phosphohexose isomerase provides an excellent example of acid-base catalysis, which involves the reversible
protonation and deprotonation of the substrate. The substrate’s hemiacetal bond is opened by the concerted action
of a protonated histidine and of a hydroxide ion, which at the outset is bound to a lysine residue. Then, a glutamate
residue abstracts a proton from C2 and donates it back to C1, which causes the C–– O double bond to migrate from
C1 to C2.

Overview of all steps of glycolysis
Step Reaction Name enzyme Class
nr.
1 Glu +ATP glu-6-P + ADP + H+ hexoKINASE transferase
2 Glu-6-P ↔ fruc-6-P phosphohexose ISOMERASE isomerase
3 fruc-6-P + ATP  fruc-1,6-diP + ADP+ H+ phosphofructo KINASE transferase
4 Fruct-1,6-diP ↔ DHAP + gly-3-P ALDOLASE lyases
5 DHAP ↔ gly-3-P triose phosphate isomerase
ISOMERASE
6 Gly-3-P + NAD+ + Pi↔ gly-1,3-diP + NADH + H+ glyceraldehyde-3-phosphate oxidoreductase
DEHYDROGENASE
7 gly-1,3-diP + ADP ↔ gly-3P + ATP phosphoglycerate KINASE transferase
8 Gly-3P ↔ gly-2P phosphoglycerate MUTASE isomerase
9 Gly-2-P ↔ PEP + H2O ENOLASE dehydratation lyase
10 PEP +ADP + H+ Pyr + ATP pyruvate KINASE transferase

Classification enzymes

Overview in metabolic pathway

Overview of other steps
There are 3 steps with a strongly negative ∆G
fermentation

• There are 3 steps with a strongly negative ∆G

• similar representation as previous slide

• There are 3 steps with a strongly negative ∆G
∆G° standard Gibbs free

energy
∆G actual Gibbs free

energy in physiological
conditions
(determined in some cell
types)

Regulation of glycolysis
• In human metabolism
• mainly by phosphofructokinase  inhibited by high
ATP levels
Phosphofructokinase in the liver is a tetramer of four

identical subunits. The positions of the catalytic and
allosteric sites are indicated.

Glycolysis: reactants and products
From minimaps

• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Fermentation

Gluconeogenesis
• Synthesis of glucose
• Glucose can be made from
noncarbohydrate precursors
• Check in pathway – large size

Gluconeogenesis
• Check in pathway – large size
• Is not a reversal of glycolysis
• In liver, brain

Gluconeogenesis
• Detail of carboxylation
Biotin-Binding Domain of Pyruvate Carboxylase. Structure of Carboxybiotin.

Regulation glycolysis
- gluconeogenesis
• Gluconeogenesis and
glycolysis
are reciprocally regulated
• Factors affecting
glycolysis – only ATP
discussed above

• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Fermentation

Glucose uptake
• Transport and utilization of glucose in the liver and in

other organs
Kinetics of glucose transport by facilitated diffusion
OUT
IN

Glucose uptake
• Transport and utilization of glucose in the liver and in

other organs
Kinetics of glucose transport by facilitated diffusion ~ Michaelis-Menten
Lowest Km – highest affinity

Glucose uptake
Via secondary transporters or secondary active transport

Glucose uptake
Uptake of glucose, galactose, and fructose in the small intestine.
sodium/glucose transporter 1 (SGLT1) is a symporter that takes up glucose (and galactose) against its concentration gradient by coupling its
transport to that of Na+

Entry points of other carbohydrates
in glycolysis
Fructose enters the glycolytic pathway in the liver

through the fructose 1-phosphate pathway

Entry points of other carbohydrates
in glycolysis
• Galactose is toxic if it can’t be converted to glucose
• Galactitol in eye diffuses to lens cataract

• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Fermentation

Fermentation or respiration
In e.g. yeast cells
In some human cells


Fermentation or aerobic oxidation

Check if reversible or irreversible reaction in next slides

Here simplification!

Alcoholic fermentation
• In e.g. yeast

Lactate fermentation
• In e.g. muscle cells

Cooperation between glycolysis
and gluconeogenesis
FERMENTATION

Cooperation between glycolysis
and gluconeogenesis

Further in this chapter

• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Fermentation

Pyruvate degradation
Major Metabolic Fates of Pyruvate and Acetyl CoA in Mammals

Pyruvate degradation
• Pyruvate transport to mitochondria
• Dehydrogenation
E1, E2, E3

• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Fermentation

Citric acid cycle – Krebs cycle –
TCA cycle (tricarboxylic acid cycle)

Citric acid cycle – Krebs cycle
Some reactions:
equilibrium reaction 
see on slide 50-51


Step Reaction Enzyme class Prosthetic group Type
*
1 Acetyl CoA + oxaloacetate + H2O → citrate + CoA + H+ Citrate synthase a
lyase
2a Citrate ⇌ cis-aconitate + H2O Aconitase lyase Fe-S b
2b cis-Aconitate+ H2O ⇌ isocitrate Aconitase lyase Fe-S c
*
3 Isocitrate + NAD+ (⇌) → α-ketoglutarate + Isocitrate dehydrogenase d + e
CO2 + NADH oxidoreductase
4 α-Ketoglutarate + NAD+ + CoA (⇌) → succinyl CoA + α-Ketoglutarate Lipoic acid, FAD, d + e
CO2 + NADH dehydrogenase complex TPP
oxidoreductase
5 Succinyl CoA + Pi + GDP ⇌ succinate + GTP + CoA Succinyl CoA synthetase f
ligase
6 Succinate + FAD (enzyme-bound) ⇌ fumarate Succinate dehydrogenase FAD, Fe-S e
+ FADH2 (enzyme-bound) oxidoreductase
7 Fumarate + H2O ⇌ L-malate Fumarase c
lyase
8 L-Malate + NAD+ ⇌ oxaloacetate + NADH + H+ Malate dehydrogenase e
oxidoreductase
Reaction type: (a) condensation; (b) dehydration; (c) hydration; (d) decarboxylation; (e) oxidation; (f) substrate-
level phosphorylation. *2 step reaction – catalysed by 1 enzyme

 Only scheme in ppt with regard

to citric acid cycle making
difference between reversible
and irreversible reactions
51 5. Metabolisme: Koolhydraatmetabolisme

see next slide
see in 2 slides

Citric acid cycle
Link between FAD and UQ

Citric acid cycle

Control of citric acid cycle

Citric acid cycle as source of
different precursors

• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Fermentation

Further in this chapter

Oxidative phosphorylation
• Oxidative phosphorylation depends on electron

transport chain (ETC) – electron transport system (ETS)
• Oxidation of NADH NAD+; FADH2 FAD
• Energetic better alternative compared to fermentation
• In humans – higher animals O2 as final e--acceptor 
aerobic respiration
• this course
• In other organisms – microorganisms: other final O 2 acceptors  anaerobic
respiration
• course microbiology

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Outer
Glycolysis
ETC Glucose
mitochondrial
membrane
Intermembrane
space
NADH ATP
Pyruvate 2*C3
Pyruvate
Oxidation Mitochondrial
matrix
NADH Acetyl-CoA CO2

2*
2*C2
NADH CO2
4*
Krebs
Cycle
FADH2 ATP
Inner
mitochondrial
e– membrane
NAD+ FAD O2 H2O ATP
e– Electron e– Chemiosmosis
Transport Chain ATP Synthase
H+

ETC
Cytosol
Outer
mitochondrial
membrane
Intra Mitochondrial
membrane matrix
space
Inner
mitochondrial
membrane

ETC

ETC
Intra-
membrane
space
Inner
mitochondrial
membrane
mitochondrial
matrix

ETC
NADH dehydrogenase Succinate dehydrogenase Q-Cytochrome C Cytochrome C oxidase

oxidoreductase
mitochondrial
matrix
Inner
mitochondrial
membrane
Intra-
membrane
space
Ubiquinone = Coenzyme Q Protein with heme group

Cytoplasmic side

ETC
Oxidant or reductant
Enzyme complex Mass Subunit Prosthetic group Matrix side Membrane Cytosolic
(kd) s core side
NADH-Q oxidoreductase 880 ≥ 34 FMN NADH Q
Fe-S
Succinate-Q reductase 140 4 FAD Succinate Q
Fe-S
Q-cytochrome c oxidoreductase 250 10 Heme bH Q Cytochrom
e c
Heme bL
Heme c1
Fe-S
Cytochrome c oxidase 160 10 Heme a Cytochrom
e c
Heme a3
CuA and CuB

ETC
Complex II: Succinate dehydrogenase
Complex I: NADH dehydrogenase

ETC
Complex III: Q-Cytochrome C oxidoreductase Complex IV: Cytochrome C oxidase

Model of proton transport
Intra membrane space
mitochondrium
matrix mitochondrium
Spring loaded valve controls the proton conduit

Receiving electrons → electron repulsion → moves the valve + proton translocated

ETC

ETC
• ETC with O2 as final electron acceptor is characteristic

for aerobic respiration
• ETC with other molecule than O2 as final electron
acceptor is characteristic for anaerobic respiration
• Is different from fermentation!
• See cursus microbiology

Reoxidation of NADH in cytosol
• During glycolyis in the cytosol, NAD+ is reduced to NADH
• This NADH is re-oxidized (to NAD+) in the cytosol because

NADH cannot pass simply the inner mitochondrial membrane.
• The “H” then transported to the mitochondria by SHUTTLES
• Example:
• malate-aspartate shuttle
• glycerophosphate shuttle

Malate-aspartate shuttle

Glycerol 3-Phosphate Shuttle

• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Fermentation

ATPase - ATPsynthase
• Proton pumping creates both a concentration gradient

and a membrane potential
• Structure of ATP synthase
• The binding-change model of ATP synthase catalysis
• ATP yield

Proton pumping creates both a concentration
gradient and a membrane potential: proton
motive force
Intramembrane
space of
mitochondrium
ETC
Matrix of
mitochondrium
After electron and proton
outer mitochondrial transport:
membrane - Free-energy of “Proton
concentration gradient”
driven H+ transport
cytosol - Free-energy of
“Electrostatic
inner mitochondrial
membrane potential”
membrane driven H+ transport

How does proton flux drive ATP
synthase?
Mitochondrial matrix
Inner mitochondrial
membrane
Intramembrane space
proton flow (between a and F0)
Please watch: https://www.youtube.com/watch?v=b_cp8MsnZFA

How does proton flux drive ATP
synthase?

Yield of ATP
Substrate level
phosphorylation
Oxidative
phosphorylation

Yield of ATP

Respiratory control

Inhibitors of electron transport
Sites of Action of Some Inhibitors of Electron Transport.

Uncoupling proteins
Action of an Uncoupling Protein. Uncoupling protein-1 (UCP-1) generates

heat by permitting the influx of protons into the mitochondria without the
synthesis of ATP.

Application: uncoupling proteins
 Used to study or treat
metabolic disorders
 Potential treatment of
cancer
Application: uncoupling proteins
Application: metabolic engineering
What kind of questions?
• Explain certain system
• E.g. shuttle system of NADH
• Classification of enzymes catalysing specific reactions
• Glycolysis: names enzymes and classes – try to see logic in the names,
make maximal use of info on pathway
• Citric acid cycle : only classes
• Fermentation versus respiration insight
• Detail working principles enzyme  when you get scheme, being able to explain
• Explain electron transport chain
•…
• Metabolic pathway can be used on exam (A3 copy provided by professor)

• No literally copying metabolic pathway as answer

• Glycolysis
• Different steps
• Regulation
• Gluconeogenesis
• Fermentation

References
• Slides prof. I. Ilsbroux
• Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York. W H
Freeman. 2002. or later edition
• Publically available LIMO, NCBI
• Chapter 16-17-18
• https://www.pearson.com/content/dam/one-dot-com/one-dot-com/us/en/higher-ed/e
n/products-services/silverthorn-7e-info/pdf/sample-chapter--
ch04.pdf
• http://watcut.uwaterloo.ca/webnotes/PDF/MetabolismNotes.pdf
• http://www.metabolic-pathway.com/fullMap.html - check browser for settings if you

have a problem – try other browser

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Metabolism:

2nd stage bachelor industrial sciences

2 5. Metabolism: Carbohydrate metabolism

3 5. Metabolism: Carbohydrate metabolism

4 5. Metabolism: Carbohydrate metabolism

5 5. Metabolism: Carbohydrate metabolism

The glycolytic pathway can be divided into three stages: (1)

6 5. Metabolism: Carbohydrate metabolism

reversible reactions + irreversible reactions → glycolysis is

ATP ADP NADH+H+ NAD+

Zie fermentatie en ETC: NADH + H+  NAD+

8 5. Metabolism: Carbohydrate metabolism

• Interactive metabolic pathways

9 5. Metabolism: Carbohydrate metabolism

The phosphate groups in ATP are

With enzyme – hexokinase - transferase

10 5. Metabolism: Carbohydrate metabolism

11 5. Metabolism: Carbohydrate metabolism

12 5. Metabolism: Carbohydrate metabolism

13 5. Metabolism: Carbohydrate metabolism

14 5. Metabolism: Carbohydrate metabolism

There are 3 steps with a strongly negative ∆G

15 5. Metabolism: Carbohydrate metabolism

• There are 3 steps with a strongly negative ∆G

16 5. Metabolism: Carbohydrate metabolism

• There are 3 steps with a strongly negative ∆G

∆G° standard Gibbs free

∆G actual Gibbs free

17 5. Metabolism: Carbohydrate metabolism

Phosphofructokinase in the liver is a tetramer of four

18 5. Metabolism: Carbohydrate metabolism

20 5. Metabolism: Carbohydrate metabolism

21 5. Metabolism: Carbohydrate metabolism

22 5. Metabolism: Carbohydrate metabolism

23 5. Metabolism: Carbohydrate metabolism

Biotin-Binding Domain of Pyruvate Carboxylase. Structure of Carboxybiotin.

24 5. Metabolism: Carbohydrate metabolism

25 5. Metabolism: Carbohydrate metabolism

26 5. Metabolism: Carbohydrate metabolism

• Transport and utilization of glucose in the liver and in

27 5. Metabolism: Carbohydrate metabolism

• Transport and utilization of glucose in the liver and in

Lowest Km – highest affinity

28 5. Metabolism: Carbohydrate metabolism

29 5. Metabolism: Carbohydrate metabolism

30 5. Metabolism: Carbohydrate metabolism

Fructose enters the glycolytic pathway in the liver

31 5. Metabolism: Carbohydrate metabolism

32 5. Metabolism: Carbohydrate metabolism

33 5. Metabolism: Carbohydrate metabolism

In e.g. yeast cells

In some human cells

34 5. Metabolism: Carbohydrate metabolism

35 5. Metabolism: Carbohydrate metabolism

36 5. Metabolism: Carbohydrate metabolism

Check if reversible or irreversible reaction in next slides

37 5. Metabolism: Carbohydrate metabolism

38 5. Metabolism: Carbohydrate metabolism

• In e.g. muscle cells

39 5. Metabolism: Carbohydrate metabolism

40 5. Metabolism: Carbohydrate metabolism

41 5. Metabolism: Carbohydrate metabolism

42 5. Metabolism: Carbohydrate metabolism