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Following Carbons Through the Cycle

To start, lets identify the carbons that initially enter the cycle
Note: G, from glucose; P, from pyruvate).

Decarboxylation during
formation of acetyl CoA
via PDH.


Enters the cycle.

As depicted below, labeling OAA with 14C in its carboxyl carbon
furthest from the keto group results in:

i) No loss of label during the decarboxylation of isocitrate to

α-ketoglutarate (via IDH).

ii) Complete loss of label upon decarboxylation of α-ketoglutarate

to succinyl CoA (via α-KG dehydrogenase) and subsequent
conversion to succinate (via succinyl CoA synthetase).

OAA Citrate Isocitrate α-KG Succinate

Thus, an apparently symmetric molecule like citrate has the potential

to react asymmetrically (i.e., it has distinguishable groups) if the
enzyme acting upon it contains an asymmetric active site.
Electrons From Cytosolic NADH Enter Mitochondria
Via Shuttles

•The inner mitochondrial membrane is impermeable to NADH and


•However, NAD+ must be regenerated from NADH in order

for glycolysis to continue.

•Shuttle systems transfer the hydrogen atom rather then

NADH itself.
The Glycerol Phosphate Shuttle
•Unidirectional; Transports reducing equivalents into mitochondria.
•Functions in skeletal muscle and brain.
Regenerates NAD+ in order to
allow glycolysis to continue.

Enzyme 2 is located on the outer surface of the inner membrane.

•FAD never penetrates the inner membrane.

•No involvement of transport proteins.

•The use of FAD rather than NAD+ enables electrons from cytosolic NADH to
be transported against an NADH gradient. The price is 1 ATP/pair of electrons.

•Thus 2 ATPS are formed per dihydroxyacetone phosphate or 4 ATPs/glucose.

The Malate-Aspartate Shuttle

•Predominantly used in heart and liver to bring electrons into

mitochondria from cytosolic NADH.

•In contrast to the glycerol phosphate shuttle which is unidirectional

and produces 2 ATPs per NADH;

•This system is bidirectional and yields 3 ATPs per NADH.

•Mediated by 2 transport proteins and 4 enzymes.

•3 ATPs are generated per NADH (or per pyruvate); 6 ATPs

per glucose.
NAD+ Feeds reducing
equivalents to
electron transport

OAA is regen-

OAA + Glu Asp + α-KG

Energy Balance Sheet
1) Glycolysis: (Glucose 2 Pyruvate)

Glucose + 2 Pi + 2 ADP + 2 NAD+

2 pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O

2) Respiration:

2 Pyruvate + 5 O2 + 30 ADP + 30 Pi
6 CO2 + 30 ATP + 34 H2O

3) Oxidation of extramitochondrial NADH during the aerobic

conversion of glucose to pyruvate (glycerol phosphate shuttle

2 NADH + 2 H+ + O2 + 4 Pi + 4 ADP
2 NAD+ + 4 ATP + 6 H2O (per glucose)

Total ATPs = 36 ATP (if use glycerol phosphate shuttle)

= 38 ATP (if use malate-aspartate shuttle)
To summarize (assuming glycerol phosphate shuttle) the overall
reaction is:

Glucose + 6 O2 + 36 Pi + 36 ADP 6 CO2 + 36 ATP + 42 H2O

ΔG˚’ = -417 kcal/mol

Separating these into their exergonic and endergonic components:

Exergonic: Glucose + 6 O2 6 CO2 + 6 H2O ΔG˚’ = -680 kcal/mol

Endergonic: 36 Pi + 36 ADP 36 ATP + 36 H2O ΔG˚’ = +263 kcal/mol

Efficiency: (263/680) (100) = 39%

The Citric Acid Cycle Provides a Source of
Biosynthetic Intermediates
In addition to serving as the major degradative pathway for ATP
generation, the cycle also serves as a major source of
biosynthetic intermediates.

1) Citrate is a carbon source

for FA and sterol biosyn.
2) AA are derived from α-KG 2
and OAA.
3) Succinyl CoA provides many
of the carbon atoms in
porphyrin synthesis.

Anaplerotic Reactions Replenish the Cycle Intermediates

•Removal of a given intermediate from the cycle will tend to slow the
flux through the cycle.

•Certain intermediates can be replenished by anaplerotic reactions.

•The principle anaplerotic reaction in humans is depicted below:

pyruvate carboxylase
Pyruvate + HCO3- + ATP Oxaloacetate + ADP + Pi

•This reaction is quite important in the liver and kidney.

•Pyruvate carboxylase is inactive in the absence of acetyl CoA, its

positive allosteric modulator.

•Thus when acetyl CoA is present in excess, it stimulates pyruvate

carboxylase to produce more OAA thereby enabling the cycle to
utilize more acetyl CoA.
Pyruvate Carboxylase and the use of Biotin to Carry CO2

Pyruvate carboxylase contains a covalently attached prosthetic group,

biotin, which carries activated CO2.

The carboxyl terminus of biotin is linked to the ε-amino group of lysine

by an amide bond.
The carboxylation of pyruvate occurs via two steps:



•In step 1, the carboxyl group is bonded to the N-1 nitrogen on the
biotin ring, thereby activating the carboxyl group.

•The activated carboxyl group is then transferred from carboxybiotin

to pyruvate to form OAA.

•The long flexible biotin-protein arm enables biotin to move from one
active site on the enzyme to the other.
Regulation of the Citric Acid Cycle

Flow of carbons entering the cycle

is regulated by PDH complex.

Regulation also occurs at the levels


citrate synthase

isocitrate dehydrogenase

α-ketoglutarate dehydrogenase