Biochemistry I, Spring Term
Lec 32: Citric Acid
Cycle & Fatty Acid
Oxidation.
Expectations:
Lecture 32
Liver Cell
O
O
Glycogen
ii) Output
Glucose
Transporter
glycogen
synthase
O
O
Gluconeogenesis
F6P
Glycolysis
iii) Location
Lactate
Transporter
Glucose
glycogen
phosphorylase
(Glu - Pyr)
Lactate
(from anerobic met)
Glucose
Hormone
Receptor
O
i) Input
April 10, 2013
(Pyr-Glu)
Triglycerides
bisPhosphatase
PFK
Fatty acids
F16P
iv) Energy Gen Steps
NAD+
NAD+
NADH
v) Regulation
NADH
Acyl-CoA
Pyruvate
Outer Mem
Mitochondria
General Features;






Inner Mem
Alanine
Acyl-CoA
O
Oxidative
Phosphorylation
ADP+Pi
2
CO2
Fatty Acid
Also known as the
Oxidation
H2O
TCA cycle
Acetyl-CoA
Amino Acids
ATP
(tricarboxylic acid) or
the Krebs cycle.
NADH
Citrate
The enzymes that
participate in the citric
FADH2
Citric Acid
acid cycle are found in
CO2
Cycle
the mitochondrial
matrix.
Catabolic role:
CO2
Amino acids, fats, and
sugars enter the TCA cycle to produce energy. Acetyl CoA is a central intermediate
Anabolic role: TCA cycle provides starting material for fats and amino acids. Note: carbohydrates cannot
be synthesized from acetyl-CoA by humans. PyruvateAcetyl CoA is one way!
In contrast to glycolysis, none of the intermediates are phosphorylated; but all are either di- or
tricarboxylic acids.
Regulation is largely by sensing energy levels.
1: Overall Carbon Flow:
All of the carbons that are input as pyruvate are released
as CO2. This is as highly oxidized as carbon can get.
Each time a CO2 is produced one NADH is produced.
This reaction is called oxidative decarboxylation.
Locations of CO2 release:
1. Pyruvate Dehydrogenase: Pyruvate to AcetylCoA
2. Isocitrate dehydrogenase: Isocitrate to ketoglutarate
3. -ketoglutarate dehydrogenase:-ketoglutarate to
succinyl-CoA
The largest change in the carbon structure occurs at step
1: The citrate synthase reaction catalyzes the following
reaction:
C2 (acetate) + C4 (oxaloacetate)  C6 (Citrate).
Subsequent reactions remove two carbons from citrate to
generate the C4 compound. Note that these carbons are not the
same as those added.
1
Pyruvate
Electron
Transport
Glucose (C6)
C3 [x2]
(Pyruvate)
CO2
C2
(Acetyl-CoA)
C4
(oxaloacetate)
C4
(succinyl
CoA)
C6
(Citrate)
CO2
C5
(ketoglutarate)
CO2
Biochemistry I, Spring Term
Lecture 32
April 10, 2013
2. Energetics of the TCA Cycle:
Glucose
In contrast to glycolysis, most of the
energetic currency is in the form of
redox reactions, only a single ATP is
produced/pyruvate while four NADH
and one FADH2 are produced. Most of
the energy from oxidation is of glucose
is harvested in the TCA cycle. The
TCA cycle is a slower but richer source
of energy.
2a: Oxidative decarboxylations:
These occur at three locations,
leading to the loss of the three
carbons from pyruvate.
1. Pyruvate dehydrogenase
2. Isocitrate dehydrogenase
3. -ketoglutarate dehydrogenase
Pyruvate dehydrogenase: This
reaction, as with α-ketoglutarate
dehydrogense, produces a thio-ester as
the oxidized species. The reaction
proceeds in two steps, loss of the CO2
group, followed by oxidation of the
aldehyde and formation of the thioester (The latter step is equivalent to
the scheme for oxidation of
glyceraldehyde-3-P in glycolysis, but
occurs by a different mechanism
here.)
The thio-ester is formed between the
oxidized product and Coenzyme A, to
form acetyl-CoA. The thio-ester is the
same oxidation state as a carboxylate.
F6P
PFK-1
bisPhosphatase-1
F 1,6 P
O
Pyr
O
H3C
O
NAD+, CoA
CO2
C
Oxaloacetate
HO
Malate
C
C
O
CoA
Citrate
CoA
O
Go=-31 kJ/mol
OH
OH
OH
C
C
Fumarate
C C COOH
C
HO
NADH
OH
O
OH
O
8 NADH =24 ATP
2 FADH2= 4 ATP
2 GTP = 2 ATP
FADH2
OH
O
S
C
C
O
O
Acetyl-CoA
C
[citrate synthase]
OH
C
H3C
C
O
O
OH
[Pyruvate
dehydrogenase]
NADH
O
O
2 ATP
2 NADH
C C COOH
OH
O
CO2
ATP
NADH
CoA
O
NADH
C
C COOH
OH
C
[a-ketoglutarate
OH
C dehydrogenase]
O
succinyl-CoA
S-CoA
O
O
a-ketoglutarate
C
O
OH
[isocitrate
dehydrogenase]
CO2
GTP
[succinate
OH
O
dehydrogenase] C
C
C
Succinate
OH iso-citrate
OH
NH2
O
CO2
O
NAD+
Pyruvate
H3C
N
O
S
H
OH
O
O
H3C
O
H3C
O
NADH
NH2
N
N
H
H
N
O
O
HS
O
N
N
H
H
OH
O
P
O
coenzymeA (CoA)
O
O
P
O
O
P
N
O
O
N
N
O
OH
O3PO
N
O
O3PO
Thioesters in Biochemical Reactions:
i) The relatively weak thioester bond facilitates the
transfer of the acetyl group to other compounds,
such as transfer of the acetyl group to
oxaloacetate in the citrate synthase reaction.
2
N
P
O
OH
O
Acetyl-CoA
N
O
O
OH
O
O
C
O C
OH
C
C
OH
O
Oxaloacetate
H3C
S
CoA
O
C
HO C
OH
C
C
H2
C
OH
O
Citrate
O
OH
Biochemistry I, Spring Term
Lecture 32
April 10, 2013
ii) Acetyl-CoA or succinyl CoA are high energy compounds, capable of driving the synthesis of GTP, such as in
the succinyl CoA synthetase in the citric acid cycle. GTP can be converted to ATP at no energy cost.
HO
O
O
P
O
O
P O
O
CH2
O
O
O
P
O
CH2
O
O
O
O
N
O
N
O
OO
P
O
NH
NH2
N
O
P O
O
O
O
O
P
O
O
N
NH2
N
OH OH
CH2
O
O
FADH2
O
OH
CH3
N
H
N
CH3
CH3
N
N
H
COH
COH
COH
CH2
O
O P
NH2
O
O
N
N
O P H
O O 2
C O N
N
O
H
H
O
OH
Succinate
N
HN
COH
COH
COH
2e- + 2 H+
CH2
O
O P
NH2
O
O
N
N
O P H
O O 2
C O N
N
REDOX
HH
O
CH3
N
HN
1. Oxidation of succinate to fumarate reduces FAD to FADH2.
Alkane → Alkene
FAD
Succinate
GTP
Alkane → Alkene → Alcohol → Ketone
OH
OH
OH OH
GDP
REDOX
O
CH2
O
2b. The remaining section of the pathway, from succinate to
oxaloacetate follows a classic three step oxidation scheme that is
also seen in fatty acid oxidation:
HH
HO
NH
S-CoA
Succinyl-CoA
O
N
HO
O
OH
Fumarate
OR
OR
HO
FADH2
(Reduced)
FAD
(Oxidized)
2. Addition of water to the double bond, to make the alcohol. Alkene → Alcohol
O
OH
O
H2O
H
H
Glucose
OH
H
H
HO
PFK-1
Fructose-1,6-P
O
OH
Malate
O
OH
Fumarate
Fructose-6-P
bisPhosphatase-1
O
Pyr
+
3. Oxidation of Malate to Oxaloacetate reduces NAD to
NADH. Alcohol → Ketone
O
OH
NAD+
NADH
O
CO2
O
CH2
Oxaloacetate
O
O
OH
Malate
[Pyruvate
dehydrogenase]
NADH
OH
O
CH2
O
NAD+, CoA
H
HO
O
H3C
O
OH
Oxaloacetate
C
C
O
OH
O Acetyl-CoA
C
CoA
H3C
S
CoA
O
C
OH
[Citrate
synthase]
HO
OH
C C COOH
C
Citrate
O
3. Regulation of the TCA Cycle:
OH
COOH
1. High energy, irreversible steps are regulated.
Succinyl-CoA
2. Regulated reactions are at the "top" of the pathway.
Examples of:
1. Product Inhibition.
2. Allosteric inhibition by feedback inhibition by products 'down stream' in the pathway.
Energy sensing is the most important regulatory control of the TCA cycle (I=inhibited)
High Energy
OTHER
Step
NADH ATP
Compound
Prod. Inh Feedback Inh
Pyruvate
I
I
 Inhibited by Acetyl CoDehydrogenase
A
Citrate Synthase
I
I
 Inh. by succinyl-CoA
CH2
CH2
O

S-CoA
Inhibited by citrate
Role of citrate in regulation of glycolysis: Citrate stabilizes the tense-form of PFK, shutting down glycolysis.
3
Biochemistry I, Spring Term
Lecture 32
April 10, 2013
Fatty acids
Fatty Acid Oxidation (-Oxidation):
A. Formation of Acyl-CoA (Cytosol): The fatty acids in the
Acyl-CoA
cytosol are coupled to coenzyme A to form acyl-CoA. The
activation reaction is catalyzed by acyl-CoA synthetase and
involves the hydrolysis of ATP to AMP, i.e. the equivalent of two
high energy ATP molecules (60 kJ/mol). The released
pyrophosphate is hydrolyzed to inorganic phosphate, making the
overall ΔG negative for the rection (indirect coupling) Note: it is
only necessary to utilize ATP once in the activation of the fatty
acid.
O
O
O
O
HS
O
O
P
N
N
O
O
P
O
Fatty Acid
activation
electron
transport
Acyl-CoA
Pyruvate
CO2
Fatty Acid
Oxidation
Acetyl-CoA
FADH2
NADH
Citrate
Citric Acid
Cycle
Adenine
CO2
O
O
OH
H
H
O
triglycerides
CO2
CoA
S
ATP
AMP
O
O
P
O
Acyl-CoA: Length N
O
P
H2O
H O
H
O
O
O
2ATP
2ADP
O
OH
O3PO
O
O
O
H3C
O
P
S
H
H
O
FAD
O
O P
O
B. Transport into mitochondria: The
H
acyl-CoA is transported into the
mitochondrial matrix for oxidation. This
location is ideal for funneling the products of oxidation (NADH and FADH2) to E. transport.
H3C
FADH2
O
CoA
S
H
H2O
C. -Oxidation (Mito. matrix):
Alkane → Alkene → Alcohol → Ketone
REDOX
OH O
H3C
S
REDOX
O
O
P
O
O
HO
CoA
Additional
Cycles
NAD+
O
Adenine
CoA
O
P
O
O
O
O
OH
N
N
H
H
O

Coenzyme A
O3PO
NADH
SH
O
H3C
Acyl-CoA is shortened 2 carbons at a time from the
carboxyl end of the fatty acid using the following
steps:
S
CoA
O
N-2 carbons
(shrinking
H3C
chain)
O
CoA
1. Formation of trans - double
+ HC
S
S
3
bond by acyl-CoA dehydrogenase, an
FAD enzyme.
2. Addition of water to the newly formed double bond to generate the alcohol by enoyl-CoA hydratase
3. Oxidation of the alcohol by NAD+ to give the ketone, catalyzed by 3-L-hydroxyacyl-CoA dehydrogenase.
4. Cleavage reaction by -ketoacy-CoA thiolase (thiolysis), generates acetyl-CoA and an acyl-CoA that is
two carbons shorter. The acetyl-CoA enters the TCA cycle.
5. Steps 1-4 are repeated until only acetyl-CoA remains.
H3C
H3C
O
O
S
CoA
H3C
CoA
4
O
O
Oxidation of C6
Fatty Acid

CoA
S

O
S
CoA
CoA
H3C
H3C
O
O
S
CoA
H3C
CoA
O
S
CoA
O
S
CoA
H3C
S
CoA
Biochemistry I, Spring Term
5
Lecture 32
April 10, 2013