CHEM 332 Krebs Cycle F2023 Handout

1/26/23
Chapter 16 - Citric Acid Cycle

TCA (tricarboxylic acid cycle)
Citric acid cycle and Krebs cycle.
Named after Sir Hans Krebs,
Nobel Laureate. He worked as
an assistant professor for Otto
Warburg (Nobel Prize 1931) and
his position terminated 1933 and
at, Sir Fredrick Gowland Hopkin's
(Nobel prize 1929) request he left
Germany to hold a Rockefeller
Studentship at the School of
Biochemistry, Cambridge. In 1953
he earned the Nobel Laureate in
Medicine for his discovery of the
citric acid cycle
The Krebs cycle is a central

pathway for recovering energy
from three major metabolites:
carbohydrates, fatty acids, and
amino acids.
Most enter the cycle through

Acetyl~CoA. The two carbons
entered at this step are lost as
CO2 (the reason you breath out
CO2). The carbon atoms that
enter by A-CoA leave after the
second turn of the cycle.
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Synthesis of the TCA

• Reactions take place in mitochondria - thus transport
of reactants and products are important
• Overall reaction involves the entry of a 2 carbon

compound (acetyl CoA) into the cycle with the loss
of 2 CO2 and formation of 3 NADH, FADH2 and GTP or
ATP.
Acetyl CoA + 2 O2 +12 ADP + 12 Pi -> 2 CO2 +CoASH +12 ATP + H2O
• No net change in the concentration of the 4 carbon

compound oxaloacetate.
• The carbons lost as CO2 are from previous A-CoAs

not from the reactant A-Co
Think of why this is a cycle vs.

pathway - not because it is written
that way.
- Oxaloacetate - only a small amount is
needed - catalytic role
- Anapleurotic - “filling up” cycle can be
used as entry and exit for production of
other essential metabolites
The TCA
• Takes place in mitochondria in the
matrix
• Like glycolysis pathway, the TCA is
highly regulated
• Sources of Acetyl CoA (another cross
road metabolite)
- glycolysis - via PDH
• ß oxidation of fatty acids
• selected amino acids
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Free energy considerations of the Citric Acid Cycle
The rxns with –DG directionally drive the pathway.

As these rxns are thermodynamically favorable, they
MUST be regulated.
Regulation of the Citric Acid Cycle
Cycle enzymes
Citrate synthase
inhibited by S-CoA,
citrate, NADH, and ATP
Isocitrate dehydrogenase
inhibited by NADH &
ATP
stimulated by NAD+ &
ADP
a-ketoglutarate
dehydrogenase
Inhibited by S-CoA,
NADH and high
ATP/AMP ratio
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Amphibolic functions of the Citric Acid Cycle Anaplerotic flux must

balance cataplerotic
① Amphibolic: flux in order to retain
Biochemical pathway
homeostasis of cellular
that involves both ***
catabolism (break metabolism.
down of molecules)
and anabolism
(construction of
molecules).
② Red arrows:
Intermediates drawn
off by cataplerotic
reactions (extract
intermediates for
biosynthesis) for use
in anabolic pathways.
③ Green arrows:
Anaplerotic reactions
replenish cycle 1) Addition of amino acids
intermediates. via transamination and
oxidative deamination
2) Odd chain fatty acids add
3) Citrate can be
transported into the
cytosol to be converted
to FA and cholesterol
Getting there - don’t forget PC!

Pyruvate Dehydrogenase (PDH) - Entry of glucose metabolites into
cycle is through formation of acetyl-CoA by oxidative
decarboxylation of pyruvate
- In eukaryotes, all of the TCA enzymes and the PDH are found in
the mitochondria. Either in the inner compartment or the matrix
of the mitochondrion.
- Pyruvate is made in the cytosol and transported by a H+ /
pyruvate symporter.
COO- NAD+ NADH

I
C=O CH3-CO ~S -CoA
I
CH3 Acetyl CoA
CoA -SH CO2
Pyruvate
Pyruvate Dehydrogenase
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PDH Exists as large multiunit complex

§ Coenzymes - Vitamin B1- thiamine pyrophosphate (TPP), panthanoic
acid (CoA), riboflavin (FAD), Niacin (NAD+) and lipoamide
• (3 different subunits)
E1 Pyruvate Dehydrogenase - 24 copies / E2 Dihydrolipoyl Translacetylase -
24 copies / E3 Dihydrolipoyl Dehydrogenase - 12 copies
- increases local concentration of substrate for each subunit
- multi-enz complexes allows little chance for
diffusion and side reactions and
direct transfer of substrate from E1 to E2 to E3
The three enzymes of the PDH

E1 - Pyruvate dehydrogenase
E2 Dihydrolipoyl Transacylase
E3 - Dihydrolipoyl Dehydrogenase
E3
E1 E2
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Reaction of the cycle

Citrate Synthase (CS) - catalyzes the condensation of acetyl-CoA
and OAA in a highly exergonic fashion. There is a substantial
conformational change in the enzyme when substrate binds.
“hides” water from the active site and then forms A-CoA
binding site. - ordered sequential reaction
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Citrate Synthase (CS)

-aldol condensation of Acetyl CoA and oxaloacetate
-involves two His and one Asp
- ordered reaction leading to tertiary changes
- induced fit caused by OAA binding forms reactive site
*
O .
O
CH3 - C ~ S- CoA
*
.
Acetyl CoA CH2 - COO-
C - COO- HO - C - COO-
CH2 - COO-
H2O CH2 - COO-
Oxaloacetate HS- CoA
Citrate
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Aconitase - catalyzes the isomeration of citrate to

isocitrate via steriospecific dehydration and
rehydration. (a two step reaction).
• citrate -> isocitrate
• isomerization reaction
• 2 steps removal and addition of water
• Iron required - not heme
• iron-sulfur protein - bound by cys residues
CH2 - COO- CH2 - COO- CH2 - COO-

HO - C - COO- C - COO- CH - COO-
CH2 - COO- H2O CH - COO
-
H2O HO-CH - COO-
Citrate Cis-Aconitate Isocitrate
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Isocitrate Dehydrogenase (IDH) - catalyzes the

oxidative decarboxylation of isocitrate to produce an a-
ketoglutarate and the first loss of CO2 – remember that to get
from citrate to OAA, you need to lose two carbons. This step
also produces NADH.
• isocitrate -> [oxalosuccinate] -> a ketoglutarate
• first oxidative conversion
• 2 steps - all on same enzyme
1 - oxidation of alcohol to ketone
NAD+ -> NADH (reduced) worth 3 ATPs
2 - ß decarboxylation
. *
.
CH2 - COO - *
CH2 - COO- CH2 - COO-
CH - COO- CH - COO- CH2

- NAD+ NADH - CO2
HO-CH - COO O C - COO O C - COO-
Isocitrate Oxalosuccinate a-Ketoglutarate
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Alpha Ketoglutarate Dehydrogenase (a KGDH)

- another enzyme multienzyme complex similar to the PDH
complex. aKGDH catalyzes the second oxidative
decarboxylation. Note that the two carbons released as CO2 in
this round of the cycle are not the carbons that entered the
cycle as acetyl-CoA
• generates CO2, NADH and succinyl CoA

• analogous to PDH
• includes the same E3 complex

NAD+ NADH
CH2 CH2
O C - COO- CO2 CoA -SH O C ~ S - CoA

a-Ketoglutarate Succinyl CoA
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Succinate Thiokinase (STK) - AKA Succinyl-CoA

synthetase – The GTP is easily converted to ATP by NDPK. In
some tissues and specific species this is an ATP specific enzyme
• succinyl CoA + GDP -> succinate + GTP + CoASH
• substrate level phosphorylation
• Hydrolysis of thioester bond of succinyl CoA -delta Go’
• GTP used by G proteins or converted to ATP by NDPK
• two forms exist - in birds ATP is produced
*
.
CH2 - COO-
ADP + Pi ATP *
.
CH2
*
.
CH2 - COO-
O C ~ S - CoA CH2 - COO-

CoA -SH
Succinyl CoA Succinate
Succinate is symmetric and SDH does not differentiate between

The COOH groups, all four carbons are labeled after one turn
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Succinate Dehydrogenase (SDH)

• Succinate + FAD -> fumarate + FADH2
• only non-matrix enzyme
• found bound to inner mitochondrial membrane
• facilitates transfer of FADH2 electrons to electron transport
system
• iron sulfur center
• FAD generally acts to oxidize C-C to C=C
• While NAD+ -> C-OH to C=O (aldehydes or ketones)
• FAD is covalently bound to protein - consequence?
CH2 - COO- FAD FADH2 CH - COO-

CH2 - COO- CH - COO-
Succinate Fumarate
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Fumarase
• fumarate -> malate
• specific addition of H2O
CH2 - COO- H2O HO - CH - COO-

Fumarate Malate
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Malate Dehydrogenase (MDH)

• malate + NAD+ -> malate + NADH
• oxidation to reform oxaloacetate
O
- NAD+ NADH
HO - CH - COO
C - COO-
-
CH2 - COO CH2 - COO-
Malate Oxaloacetate
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Regulation of PDH and the cycle

Regulated to meet needs of cell - don’t waste energy
1 Feed back inhibition at PDH
– ACoA and NADH are allosteric inhibitors
• Act by inhibiting E2 - shifting the equilibrium twords
the acylated form. Leads to TTP build up and will
decrease decarboxylation
( -) ( -)
COO- NAD+ NADH
I
C=O CH3-CO ~S -CoA
I
CH3 Acetyl CoA
CoA -SH CO2
Pyruvate
Pyruvate Dehydrogenase
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Mitochondria
- Outer membrane is very porous
- Inner membrane very tight. Transfer into and out
of matrix is controlled - important in H+ and
shuttling reducing equivalents.
- membranes are
topologically sided -
different charge,
lipids and proteins
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1/26/23

Chapter 16 - Citric Acid Cycle

The Krebs cycle is a central

Most enter the cycle through

Synthesis of the TCA

• Overall reaction involves the entry of a 2 carbon

• No net change in the concentration of the 4 carbon

• The carbons lost as CO2 are from previous A-CoAs

Think of why this is a cycle vs.

Free energy considerations of the Citric Acid Cycle

The rxns with –DG directionally drive the pathway.

Regulation of the Citric Acid Cycle

Amphibolic functions of the Citric Acid Cycle Anaplerotic flux must

Getting there - don’t forget PC!

COO- NAD+ NADH

PDH Exists as large multiunit complex

The three enzymes of the PDH

Reaction of the cycle

Citrate Synthase (CS)

Aconitase - catalyzes the isomeration of citrate to

CH2 - COO- CH2 - COO- CH2 - COO-

Isocitrate Dehydrogenase (IDH) - catalyzes the

CH - COO- CH - COO- CH2

Alpha Ketoglutarate Dehydrogenase (a KGDH)

• generates CO2, NADH and succinyl CoA

CH2 - COO- CH2 - COO-

O C - COO- CO2 CoA -SH O C ~ S - CoA

Succinate Thiokinase (STK) - AKA Succinyl-CoA

O C ~ S - CoA CH2 - COO-

Succinate is symmetric and SDH does not differentiate between

Succinate Dehydrogenase (SDH)

CH2 - COO- FAD FADH2 CH - COO-

CH2 - COO- H2O HO - CH - COO-

CH2 - COO- CH2 - COO-

Malate Dehydrogenase (MDH)

Regulation of PDH and the cycle

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