Aerobic Metabolism of
carbohydrate
The three stages of respiration
• Stage I All the fuel molecules are oxidized to
acetyl-CoA.
• Stage II The acetyl-CoA is completely oxidized
into CO2, electrons were collected by NAD and
FAD via the citric acid cycle.
• Stage III Passage of electrons through the
electron transport system to yield ATP from
oxidative phosphorylation.
Pyruvate Oxidation
Pyruvate + CoA + NAD+
acetylCoA + CO2 + NADH + H+
Pyruvate Dehydrogenase is a large
complex:
pyruvate dehydrogenase (E1),
dihydrolipoyl transacetylase (E2),
dihydrolipoyl dehydrogenase (E3)
Requires 5 coenzymes:
TPP, Lipoic Acid, Coenzyme A, FAD,
NAD+
Reactions of the PDH complex
Regulation of Pyruvate
Dehydrogenase
1). Product inhibition by NADH & acetyl CoA
2). Covalent modification
Citric Acid Cycle
• Hans Krebs proposed the “citric acid
cycle” for the complete oxidation of
pyruvate in animal tissues in 1937
(1953 Nobel Prize laureate).
• The tricaboxylic acid (TCA) Cycle,
Krebs Cycle
Citric Acid Cycle
• The common pathway leading to
complete oxidation of carbohydrates,
fatty acids, and amino acids to CO2.
• Some ATP is produced, More NADH is
made ,NADH goes on to make more ATP
in electron transport and oxidative
phosphorylation
• A pathway providing many precursors for
biosynthesis
citric acid cycle overview
Individual reaction
Reaction 1: Citrate Synthase
Allosteric,
-ATP,NADH,
succinyl-CoA
A thioester: so a high
energy compound
Hydrolysis helps drive
this reaction forward
Fluoroacetate blocks the cycle
• Fluoroacetate is poisonous because it
can convert to fluorocitrate which is an
inhibitors of TCA cycle.
Reaction 2:
Aconitase
Dehydration followed
by hydration
Reaction 3:
Isocitrate
Dehydrogenase
First Oxidative
decarboxylation
Allosteric enzyme
-ATP,NADH
Ketoglutarate
Dehydrogenas
e
Second Oxidative
decarboxylation
Ketoglutarate dehydrogenase complex is
very similar to the pyruvate degydrogenase
complex.
allosteric inhibitor: NADH,succinylCoA,ATP
• Five coenzymes used –
• TPP, CoASH, Lipoic acid,NAD+, FAD
Reaction 5: Succinate
thiokinase
(also called succinyl CoA
synthetase)
Hydrolysis
Substrate Level
Phosphorylation
A thioester: so
a high energy
cpd
Reaction 6:
Succinate
Dehydrogenas
e
2
Oxidation
e- carrier is FAD
Succinate Dehydrogenase
• Part of electron transport chain in the
inner membrane of mitochondria.
• Removal of H across a C-C bond is not
sufficiently exergonic to reduce
NAD+,but it does yield enough energy
to reduce FAD.
• Malonate is a competitive inhibitor
Reaction 7:
Fumarase
Hydration
trans-addition of the
elements of water across the
double bond, forms L-malate
Reaction 8: Malate
Dehydrogenase
Oxidation
This and the
previous two
reactions form a
reaction triad
+
TCA Cycle Summary
1 acetate
through the
cycle
produces 2
CO2, 1 GTP,
3NADH,
1FADH2
Aerobic Nature of the Cycle
NADH and FADH2 must be reoxidized by
the electron transport chain.
Succinate Dehydrogenase is part of
electron transport chain in the inner
membrane of mitochondria.
Energetics
• Energy is conserved in the reduced
coenzymes NADH, FADH2 and one
GTP
• NADH, FADH2 can be oxidized to
produce ATP by oxidative
phosphorylation
1.5
1.5
7.5
ETS
1.5
7.5
7.5
ATP generated by the cycle
3 NAD+
3 NADH
ETS
3*2.5=7.5 ATP
FADH2
FAD
ETS
1.5 ATP
Substrate level
phosphorylation
1 GTP
10 ATP
Equivalents
Total
ATP generated by complete oxidation of
Glucose
glucose
glycolysis
2ATP(Substrate-level phosphorylation) 2
2NADH ( oxphos) 3-5
2Pyruvate
oxidative decarboxylation
2 NADH
5-7ATP
( oxphos) 5
2 Acetyl CoA
TCA cycle 20
6 NADH
2 FADH2
2 GTP
total 30-32ATP
CO2
25ATP
The Fate of Carbon in TCA
Always the 2
carbons from
acetyl CoA
Reaction 2:
Aconitase
Stereospecific
A symmetrical compound
is converted to a chiral
compound
How much
radioactivity has
been lost after
one turn?
*
*
**
**
**
**
Zero
Stereospecifi
c
**
* * Not
* * stereospecific
**
Not stereospecific
**
**
**
How much
radioactivity
has been
lost after 2
turns?
50%
****
**
**
**
**
**
**
**
Cycle 2
**
**
**
*
**
*
*
*
**
*
• Carboxyl C of acetate turns to CO2 only
in the second turn of the cycle
• Methyl C of acetate survives two cycles
completely, but half of what's left exits
the cycle on each turn after that.
Regulation of the TCA Cycle
Again, 3 irreversible reactions are the key
sites
• Citrate synthase - regulated by availability
of substrates - acetyl-CoA and
oxaloacetate, citrate is a competitive
inhibitor;
Allosteric: - NADH , ATP,succinyl-CoA
• Isocitrate dehydrogenase – NADH,ATP
inhibit, ADP and NAD+ Ca++ activate
• -Ketoglutarate dehydrogenase - NADH
and succinyl-CoA inhibit, AMP Ca++activate
Major regulatory
sites are
irreversible
reactions
Anaplerotic reactions
• Anaplerotic (filling up) reactions replenish
citric acid cycle intermediates
• Amphibolic Nature of TCA Cycle means
it both Anabolic and Catabolic. TCA
cycle provides several of Intermediates
for Biosynthesis
Anaplerotic reactions
• PEP carboxylase - converts PEP to
oxaloacetate , Anaplerotic reaction in
plants and bacteria
• Pyruvate carboxylase - converts
pyruvate to oxaloacetate, a major
anaplerotic reaction in mammalian tissues
• Malic enzyme converts pyruvate into
malate
The Glyoxylate Cycle
•
•
•
•
An Anabolic Variant of the Citric Acid Cycle
for plants and bacteria
Acetate-based growth - net synthesis of
carbohydrates and other intermediates from
acetate - is not possible with TCA
Glyoxylate cycle offers a solution for plants
and some bacteria and algae
The CO2-evolving steps are bypassed and
an extra acetate is utilized
Isocitrate lyase and malate synthase are the
short-circuiting enzymes
Acetyl-CoA
Citrate
Oxaloacetate
Isocitrate
Malate Malate sythase
Glyoxalate
Acetyl-CoA
Isocitrate
lyase
Succinate
• 在肌肉糜中加入柠檬酸可刺激氧的消耗
，而加入丙二酸后呼吸被抑制，并造成
琥珀酸的积累，请解释这现象，有什么
办法可以去除丙二酸的抑制。
• 糖酵解可以在有氧和无氧的情况下发生
，而TCA循环却只能在有氧情况下发生
。解释为什么糖酵解可以在无氧情况下
进行而TCA循环却不能。TCA循环中最
直接受到无氧影响的是哪些步？
Home work
• Starting with pyruvate labelled with 13C on
the keto-carbon, where will this isotopic
label end up in oxaloacetate after one
round of the citric acid cycle?
Briefly describe
• The reaction catalyzed by the pyruvate
dehydrogenase complex in
carbohydrate metabolism:
• One advantage of a multienzyme
complex
• explain how pyruvate dehydrogenase is
regulated. (Graphs of enzyme activity
would be cool)!
• What effect would increasing
expression of the PDH kinase gene
have on carbohydrate metabolism?
• If you add pyruvate labeled in the
carboxylate group to actively metabolizing
eukaryotic cells under aerobic conditions,
under what conditions will that labeled
carbon enter the citric acid cycle?
• What is the primary regulatory site in
glycolysis? Explain the role of energy
charge in controlling the enzyme.
• Name other sites for regulation in
glycolysis.