Biochemistry I
Lecture 32: Citric Acid
Cycle & Fatty Acid
Metabolism.
Lecture 32
Liver Cell
Hormone
Receptor
November 15, 2015
Glucose
Glucose
Transporter
Expectations for Citric
Acid Cycle:
glycogen
Glucose
synthase
i) Input - pyruvate
Glucoglycogen
ii) Output CO2, NADH,
neogenesis
Glycogen
phosphorylase
(Pyr->Glu)
FADH2, GTP
F6P
Triglycerides
Glycolysis
iii) Location
bis(Glu -> Pyr) PFK
Phosphatase
mitochondrial
Fatty acids
F16P
matrix
NAD+
iv) Energy Generating
NADH
Acetyl-CoA
Stepsoxidative
Acyl-CoA
ATP Pyruvate
decarboxylations
Oxidative
Outer Mem
v) Regulation – energy
Electron PhosInner
Mem
Transport phorylation
sensing (NADH, ATP). Mitochondria
Acyl-CoA
Alanine
Pyruvate
vi) Biosynthesis of amino
ADP+Pi
O2
CO2
Fatty Acid
acids.
Oxidation
H2O
Acetyl-CoA
Amino Acids
Features of Citric Acid
ATP
Cycle:
NADH
Citrate
 Also known as the
TCA cycle
FADH 2
Citric
acidAcid
(tricarboxylic acid)
Citric
cycle (TCA, Krebs)
CO2
Cycle
or the Krebs cycle.
 The enzymes that
participate in the
CO2
citric acid cycle are
found in the mitochondrial matrix.
 Catabolic role: 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:
 Pyruvate Dehydrogenase: Pyruvate to acetylCoA
 Isocitrate dehydrogenase: Isocitrate to ketoglutarate
 -ketoglutarate dehydrogenase:-ketoglutarate
to succinyl-CoA
The largest change in the carbon structure occurs at
step 1, the citrate synthase reaction:
C2 (acetate) + C4 (oxaloacetate)  C6 (Citrate)
Subsequent reactions remove two carbons from citrate
to generate the C4 compound, oxaloacetate at the end
of the cycle.
1
Glucose (C6)
C3 [x2]
(Pyruvate)
CO2
C2
(Acetyl-CoA)
C4
(oxaloacetate)
C4
(succinyl
CoA)
C6
(Citrate)
CO2
C5
(ketoglutarate)
CO2
Biochemistry I
Lecture 32
2. Energetics of the TCA Cycle:
 Most of the energetic currency is
in the form of redox reactions, only
a single ATP (GTP) 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 (step 0)
2. Isocitrate dehydrogenase (step 3)
3. -ketoglutarate dehydrogenase
(step 4)
Pyruvate dehydrogenase
(decarboxylase)(Step 0)
1. loss of the CO2 group.
2. oxidation of the aldehyde and
formation of the thio-ester. (The
thio-ester is the same oxidation
state as a carboxylate.)
The thio-ester is formed between
the oxidized product and
Coenzyme A, to form acetyl-CoA.
November 15, 2015
Glucose
F6P
bisPhosphatase-1
PFK-1
F 1,6 P
2 ATP
2 NADH
Pyr
NAD+, CoA
CO2
NADH
Oxaloacetate
[Pyruvate 0
dehydrogenase]
Acetyl-CoA
Citrate
CoA
8
[citrate synthase] 1
Go=-31 kJ/mol
2
NADH
Malate
iso-citrate
8 NADH =24 ATP
2 FADH2= 4 ATP
2 GTP = 2 ATP
7
Fumarate
FADH2
[succinate 6
dehydrogenase]
ATP
GTP
NADH
[isocitrate 3
dehydrogenase]
NADH
CoA
4
Succinate
[a-ketoglutarate
dehydrogenase]
a-ketoglutarate
[succinate 5
thiokinase]
succinyl-CoA
NADH
CO2
NAD+
Pyruvate
Acetyl-CoA
coenzymeA (CoA)
Thioesters in Biochemical Reactions: The relatively weak
thioester bond facilitates the transfer of the attached group to
other compounds.
i) Citrate synthase mechanism (Step 1).
Asp-375 – general base
His-274 – general acid
A) proton abstraction by Asp375, proton donation by His274
B) nucleophilic attack of –ene to C=O on oxaloacetate.
C) hydrolysis of thioester.
A
Oxaloacetate
Citrate
B
C
1
3
3
2
2
1
2
HS-CoA
Biochemistry I
Lecture 32
A
B
November 15, 2015
C
Succinate
ii) Succinate thiokinase
(Step 5): succinyl CoA
can provide enough
energy to driving the
synthesis of GTP.
GTP
A) phosphorlysis of thioSuccinyl-CoA
CoA ester.
B) Transfer of phosphate
to His
C) Transfer from
phosphoryl-His to GDP, forming GTP.
S-CoA
GDP
2b. The remaining section of the pathway, from succinate to
oxaloacetate follows a classic three step oxidation scheme
(also seen in fatty acid oxidation):
Alkane → Alkene → Alcohol → Ketone
REDOX
REDOX
Step 6. Oxidation of succinate to fumarate reduces FAD to
FADH2. Alkane → Alkene
O
FAD
OH
O
O
O
CH3
N
HN
CH3
N
N
OH
O
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
H
H
OR
HO
O
OH
Succinate
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
FADH2
HH
HH
O
O
OH
Fumarate
HO
Step 7. Addition of water to the double bond, to make the
alcohol. Alkene → Alcohol
OR
FADH2
(Reduced)
FAD
(Oxidized)
Glucose
PFK-1
Fructose-6-P
bisPhosphatase-1
Fructose-1,6-P
Malate
Fumarate
O
OH
NAD+
NADH
O
CO2
O
[Pyruvate
dehydrogenase]
NAD+, CoA
OH
NADH
CH2
O
O
H3C
H
HO
O
Pyr
Step 8. Oxidation of Malate to Oxaloacetate
reduces NAD+ to NADH. Alcohol → Ketone
O
OH
CH2
O
O
OH
Oxaloacetate
Malate
Oxaloacetate
O
C
C
O
OH
O Acetyl-CoA
C
CoA
H3C
S
CoA
O
C
OH
[Citrate
synthase]
HO
OH
C C COOH
C
Citrate
Regulation of the TCA Cycle:
O
OH
1. High energy, irreversible steps are regulated.
2. Regulated reactions are at the "top" of the
Succinyl-CoA
pathway.
Examples of:
1. Product Inhibition.
2. Allosteric inhibition by feedback inhibition by products 'downstream' in the pathway.
Energy sensing is the most important regulatory control of the TCA cycle (I=inhibited)
High Energy
OTHER
Step
NADH ATP
Compound
Product Inh Feedback Inh
I
I
Pyruvate
Inhibited by Acetyl Co-A
Dehydrogenase
COOH
CH2
CH2
O
Citrate Synthase
I
I
S-CoA
Inhibited by succinyl-CoA
Inhibited by citrate
Regulation of glycolysis: Citrate stabilizes the tense-form of PFK, shutting down glycolysis.
3
Biochemistry I
Lecture 32
November 15, 2015
Fatty Acid Oxidation (-Oxidation):
A. Formation of
Acyl-CoA: Length N
FAD
1
FADH2
AMP
2ATP
2ADP
2
H2O
Acyl-CoA
(Cytosol):
Additional
The fatty acids in the
Cycles 5
NAD+
cytosol are coupled
3
to coenzyme A to
NADH
form acyl-CoA. The
activation reaction is
catalyzed by acylCoenzyme A

CoA synthetase and
4
involves the
hydrolysis of ATP to
AMP, i.e. the
N-2 carbons
equivalent of two
(shrinking
chain)
high energy ATP
molecules (60 kJ/mol). The released pyrophosphate is hydrolyzed to inorganic phosphate, making
the overall ΔG negative for the reaction (indirect coupling).
Note: it is only necessary to utilize ATP once in the activation of the fatty acid.
B. Transport into mitochondria: The acyl-CoA is transported into the mitochondrial matrix -ideal
for funneling the products of -oxidation (NADH and FADH2) to E. transport.
C. -Oxidation (Mito. matrix): Acyl-CoA is shortened 2 carbons at a time from the carboxyl end of
the fatty acid using the following steps:
1. Formation of trans - double 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 acylCoA that is two carbons shorter. The acetyl-CoA enters the TCA cycle.
5. Steps 1-4 are repeated until only acetyl-CoA remains. The last cycle produces two acetylCoA.

H3C
O
O
Oxidation of C6
Fatty Acid
H3C
O
O
S
CoA
H3C
CoA
S

O
S
CoA
CoA
H3C
H3C
O
O
S
CoA
O
CoA
O
S
H3C
S
CoA
H3C
S
CoA
CoA
Fatty Acid Synthesis: Occurs in the cytosol using acetyl CoA (derived from citrate), elongating by
2 carbons at a time, each pair of Cs is added from malonyl-CoA. Electron donor is NADPH.
Malonyl-S
Acetyl-CoA
1
Acetyl-CoA
4
2
1
3
2
3