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[BIOCHEM] 2.

4 TCA/PDH/HPM/Uronic
Acid Pathway
[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic
Acid Pathways Dr. Reyes
Dr. Reyes

August 3, 2013

Beley, Bernabe, Bernaldez, Berris, Bilasano, Bilbao, Brigola


OUTLINE

I.

TRICARBOXYLIC ACID

OVERVIEW OF CITRIC ACID CYCLE

I. Tricarboxylic Acid
A. Overview of Citric Acid Cycle
B. Pathways
C. Energetics of TCA cycle
D. Summary of TCA cycle
E. Other Aspects of the cycle
F. Catabolism of the Major Nutrients Converge on
G. the TCA cycle
H. Role of Vitamins in TCA cycle
I. Amphibolic Nature of TCA cycle
J. Anaplerotic Reactions
K. Regulation of Krebs cycle
L. Clinical correlation
M. ATP Yields
N. Shuttle Mechanisms for Cytoplasmic Reducing
O. Equivalents
P. Summary of all Reactions of TCA cycle
II. Pyruvate Dehydrogenase Complex
A. General Nature of Pyruvate Dehydrogenase Complex
B. Fate of Pyruvate
C. PDH Complex Mechanism of Action
D. Pyruvate Dehydrogenase Regulation
E. Alterations in PDH Complex
III. Pentose Phosphate Pathway
A. Two Phases of Pentose Phosphate Pathway
B. Differences between Glycolysis and Pentose Phosphate Pathway
C. Biomedical Importance of Pentose Phosphate Pathway
D. Other pathways that require NADPH
IV. Uronic Acid Cycle
V. Appendix

CITRIC ACID CYCLE






Pyruvate (product of aerobic glycolysis) oxidizes


completely to CO2 and H2O via acetyl CoA through this
cycle
Occurs in the Matrix of the Mitochondria
st
Citrates, 1 substrate/compound that was studied

Other names:
A. Krebs Cycle: (after Hans Krebs), biochemist who
discovered the cycle in 1930
B. Tricarboxylic Acid Cycle (TCA): most common
name of the pathway because of the involvement of
tricarboxylates: citrate and isocitrate
Site: Mitochondrion (matrix)
*appropriate location since the 2 primary sources of acetyl
CoA: pyruvate dehydrogenase complex and fatty acid oxidation sequence, are located in the mitochondrion

OBJECTIVES
At the end of the lecture, the student should be able to:
1. Explain the Unique features and their roles in carbohydrate
metabolism: (pyruvate dehydrogenase reaction, TCA cycle,
HMP shunt, Uronic Acid Pathways)
2. Discuss the Regulation of the PDH reaction.

Major function: Final Common Pathway of all


Macronutrients (oxidation of carbohydrates, lipids, and
proteins) via the formation of a common metabolite, Acetyl
CoA.

TCA

ACETYL CoA --- COMMON METABOLITE OF OXIDATION


OF FUEL NUTRIENTS

1.
2.
3.
4.
5.
6.
7.

Explain the key role of pyruvate in central metabolism


Illustrate the general nature of the PDH reaction and its
cofactors.
Discuss the key control mechanism which regulates the
pyruvate dehydrogenase complex.
Discuss the sources and fates of Acetyl CoA in normal
metabolism.
Explain the overall process which occurs in the
tricarboxylic acid cycle
Enumerate the key intermediates in TCA cycle
Explain the concept of anaplerotic reactions

Pentose Phosphate Pathway


1. Explain the role of the Pathway in metabolism in terms of
its substrate and products
2. Enumerate key features of the HPM.
3. Discuss the essential role of the pentose phosphate
pathway in providing key biosynthesis intermediates.

1.

2.
3.

End Products: CO2 and ATP


*ATP produced here accounts for 2/3 generated from fuel
oxidation

References:
Carbohydrate Metabolism Lecture Notes by Dr. Reyes
Citric Acid Cycle PPT by Dr. Madarcos

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[Biochem]

The oxidation of glucose (via pyruvate), fatty acids (via


beta-oxidation), amino acids and ketone bodies (via
acetoacetate and beta-hydroxybutyrate) leads to the
formation of a common metabolite, acetyl coA.
Dietary acetate (acetic acid) can also be activated into
acetyl coA; hence, can also be converted into energy.
Acetyl CoA, the common point of convergence for the
major pathways of fuel oxidation, is therefore the
substrate for TCA.

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


PATHWAYS
Overview

c. Pyruvate Dehydrogenase Complex includes the ff:


o Pyruvate dehydrogenase/Thiamine pyrophosphate
(TPP) --- E1
o Dihydrolipoyl transacetylase/Lipoic acid --- E2lipoamide
o Dihydrolipoyl dehydrogenase/ FAD --- E3-FAD
d.

The 2 other coenzymes of pyruvate dehydrogenase are:


o Coenzyme A (CoA)
+
o Nicotinamide adenine dinucleotide (NAD )
+

Note: Pyruvate is transported in symport with H

CLINICAL CORRELATION
Deficiency in the E1: inability to convert pyruvate to acetyl
CoA  pyruvate shunted to lactic acid via lactate
dehydrogenase  congenital lactic acidosis  problems for
the brain that utilizes ATP from pyruvate oxidation.
2) Synthesis of Citrate from
Oxaloacetate (or Condensation)

Acetyl

CoA

and

10 Steps Pathway
1)

Oxidative Decarboxylation of Pyruvate a


complicated 5-step reaction process but the overall
reaction is as follows:

Acetyl CoA condenses with oxaloacetate and H2O


catalysed by citrate synthase to form an intermediate
citrate or citric acid plus CoA
A C-C bond is formed between the methyl carbon of
acetyl CoA and the carbonyl carbon of oxaloacetate.
Highly Exergonic
Only reaction where C-C bond forms
This is the primary pacemaker step or the primary
control point of the TCA

a.

b.

a. Pyruvate enters the mitochondrion via pyruvate


translocase (transport protein embedded in the inner
mitochondrial membrane)
Oxidative decarboxylation takes place in the
presence of CoASH (Coenzyme A) to acetyl coA
+
+
NAD accepts 2 electrons (from pyruvate); NAD is
+
+
reduced to NADH (NADH + H )
Highly exergonic and irreversible

c.
3)

Isomerization of Citrate (Rearrangement)

b. 1 Glucose = 2 pyruvate produced; hence,


2 mol of acetyl CoA
2 mol of CO2
2 NADH
1 CO2 released
Note: This reaction pathway is not part of the TCA Cycle
proper but it links the glycolytic pathway with the TCA
a.
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[Biochem]

Citrate is isomerized to isocitrate by aconitase


because it cannot be oxidized directly.

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


b.

The OH group of citrate at carbon 3 is removed as


H2O to form a C=C, followed by stereospecific
addition of H2O to the intermediate to form isocitrate,
hence isomerization involves two steps:
i. Dehydration of citrate to cis-aconitate by
aconitase.
ii. Followed by rehydration of cis-aconitate to
isocitrate again by aconitase.
Reversible reaction
Fluorocitrate a potent inhibitor of aconitase 
accumulation of citrate;
lethal in small dose and has been used as a
rat poison

c.

4)

Dihydrolipoamide dehydrogenase)

With coenzymes which include: TPP,


+
Lipoic acid, FAD, NAD and CoA

Exergonic and Irreversible


nd

Production of the 2 NADH and CO2


b.
6)

Cleavage of Succinyl CoA

Oxidation and Decarboxylation of Isocitrate

a.

a.

b.
5)

Inhibition: increase ATP, GTP, NADH, Succinyl


CoA and Arsenite

Isocitrate (decarboxylation and redox reaction)


catalyzed by Isocitrate dehydrogenase leads to the
formation of oxalosuccinate, and thereafter Ketoglutarate
2+
Decarboxylation step requires either Mg or
2+
Mn
Exergonic and Irreversible
st
Yields both 1 NADH and CO2

b.

Inhibition: increase ATP and NADH levels,


Activation: increase mitochondrial ADP levels

Oxidative Decarboxylation of -ketoglutarate

a.

The formed GTP in this process is used to


decarboxylate oxaloacetate  Phosphoenolpyruvate
(PEP) in gluconeogenesis (this provides a link between
gluconeogenesis and TCA cycle)

Kidney and Liver tissues (site of gluconeogenesis) have


2 isoenzymes of succinate thiokinase, 1 for ADP and 1
for GDP. Non-gluconeogenic tissues have only one for
ADP

Decarboxylation and redox reactions catalyzed by


the -Ketoglutarate dehydrogenase complex
(formed by -ketoglutarate dehydrogenase,
Dihydrolipoamide
succinyl
transferase,
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[Biochem]

Cleavage of Succinyl CoA (substrate-level


phosphorylation) is catalyzed by Succinyl CoA
Synthetase/Succinate thiokinase.
2+

occurs with Mg , which then aids in the


transfer of a PO4 group from GDP to form
GTP (same as the energy content of ATP)

Exergonic and Irreversible


In the catalytic mechanism of Succinate thiokinase:
o Phosphate displaces CoA from Succinyl CoA
 forms the intermediate anhydride succinyl
PO4
o Phosphoryl group is transferred to a histidine
residue found in the enzyme  formation of
phosphoenzyme intermediate
o Release of Succinyl, transfer of phosphoryl
group from phosphoenzyme intermediate to
GDP  GTP

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


7)

Oxidation of Succinate to Fumarate

8)

Hydration of Fumarate

a.





Fumarate is hydrated to L- malate by fumarase (or


fumarate hydratase)
Freely reversible reaction
H2O (with an OH and a proton) is added to the double
bond of fumarate in the trans position to form malate.
Hence an alkene (fumarate) is converted to an alcohol
(malate).

9)

Oxidation of Malate to Oxaloacetate

Malate is oxidized to oxaloacetate by malate


dehydrogenase
rd
3 and final NADH of the cycle
+
Here NAD is the acceptor of 2 hydrogen atoms and 2
electrons, forming the reduced NADH
o alcohol group of malate is oxidized to a keto group
forming oxaloacetate through the donation of
+
electrons to NAD
o Reversible
+
o However, the NADH produced by the three NAD linked dehydrogenases of the TCA cycle is rapidly
+
reoxidized to NAD by the respiratory chain, thus
favoring the forward direction of malate
dehydrogenase
The conversion of malate to oxaloacetate completes
one round trip through the TCA cycle
The oxaloacetate may then react with another molecule
of acetyl CoA continuing the cycle of reactions to
generate again oxaloacetate.
o Oxaloacetate is not really considered a substrate of
the cycle or a source of electrons or carbon
This reaction step is analogous to the lactate
dehydrogenase reaction of glycolysis

b.

c.

d.
e.

Via succinate dehydrogenase (also called Complex


II), a flavoprotein containing the prosthetic group
FAD (also contains iron-sulfur centers aside from
+
FAD), with FAD as a hydrogen acceptor,
succinate is oxidized to fumarate, forming the
reduced coenzyme FADH2.
+

C=C is formed with loss of 2 H and 2


electrons

Dehydrogenation reaction is stereospecific


 only the trans isomer of the product
fumarate is formed.

Freely reversible
+
FAD is used as a hydrogen acceptor (instead of
+
the more usual NAD )

because the free energy change is


+
insufficient to reduce NAD
+

FAD is nearly always the electron


acceptor in oxidations that remove 2
hydrogen atoms from a substrate.
Complex II in eukaryotes is embedded in the inner
mitochondrial membrane whereas the other
enzymes of the TCA are dissolved in the
mitochondrial matrix.
Hence succinate dehydrogenase is the only
enzyme of the TCA cycle that is not found in the
mitochondrial matrix.
Substrate malonate: competitive inhibitor of
succinate dehydrogenase or Complex II.
o inhibits succinate because of the very close
structural similarity between the two 
malonate is a substrate analogue.
o It is a dicarboxylic acid (similar to succinate) 
binds to cationic amino acid residues in the
active site of succinate dehydrogenase but
does not react or undergo oxidation because it
lacks the CH2-CH2 group necessary for
dehydrogenation.








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[Biochem]

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


nd

ENERGETICS OF TCA CYCLE

3.

4.

Like all metabolic pathways, the TCA cycle


operates with an overall negative G
G = -13.1 kcal/mole, acetyl CoA  oxaloacetate,
energetically favorable
reactions with large negative G values are
indicated by red arrows (in the pathways figures)
which is strongly irreversible (exergonic reactions).

5.

OTHER ASPECTS OF THE CYCLE





SUMMARY OF TCA CYCLE





Legend:
Words in Bold end-product per step
Words Underlined enzyme/s used
1.

2.

Pyruvate oxidized to acetyl CoA by pyruvate


dehydrogenase enzyme complex; this reaction links
glycolytic pathway to TCA cycle
Eight steps of TCA proper:

Acetyl CoA condenses with oxaloacetate via citrate


synthase to form citrate or citric acid; primary
control point of TCA

Citrate isomerized to isocitrate by aconitase

Isocitrate oxidatively decarboxylated to ketoglutarate


by
NAD-linked
isocitrate
st
dehydrogenase, releasing the 1 CO2 of the two
st
and the 1 of the NADH of the cycle

-ketoglutarate is oxidatively decarboxylated to


succinyl CoA by NAD-linked -ketoglutarate




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[Biochem]

dehydrogenase complex, releasing the 2 CO2 and


nd
producing the 2 NADH of the cycle.

Succinyl CoA converted to succinate by succinyl


CoA thiokinase, generating GTP (or ATP) at
substrate level

Succinate oxidized to fumarate by succinyl


dehydrogenase, a flavin enzyme, forming the
reduced FADH2

Fumarate hydrated to malate via fumarase

Malate oxidized to oxaloacetate by NAD-linked


rd
malate dehydrogenase, producing the 3 and final
NADH of the cycle
TCA operates until all acetyl CoA are oxidized

Another molecule of acetyl CoA can enter the TCA


since there are 2 pyruvates per molecule of
glucose
Inhibitors of the cycle: Fluorocitrate, Arsenite and
Malonate
Four (4) vitamins required for TCA cycle:
+

Niacin (NAD )

Pantothenic acid (CoA)

Thiamine (TPP)

Riboflavin (FAD)

TCA is cyclic while Glycolysis is linear


3 Carbon atoms enter the mitochondrion as pyruvate;
one is lost as CO2 in its conversion to acetyl CoA
Remaining 2 Carbon atoms enter TCA as acetyl CoA
which is then converted to citrate (step2)
Citrate is degraded by a sequence of reactions where 2
Carbon atoms are lost as CO2; one in isocitrate
dehydrogenase reaction and the other in ketoglutarate dehydrogenase reaction
Further turn of the cycle regenerates the 4-carbon
oxaloacetate which now combines with another
molecule of acetyl CoA to start another revolution of the
cycle
Thus, in 1 cycle:
o 1 Acetyl CoA enters
o 2 molecules of CO2 are released
o 3 NADH2 are produced
o 1 molecule of oxaloacetate is utilized to form citrate
but is regenerated at the end of the cycle
Because 2 molecules of acetyl CoA are produced from
each glucose molecule, 2 cycles are required per
glucose molecule; therefore at the end of all cycles
(products of acetyl CoA oxidation):
o 2 GTPs
o 4CO2
o 6 NADH2
o 2 oxaloacetates regenerated.
Hence, there is NO net production nor consumption
of oxaloacetate in the operation of the cycle from
incoming acetyl groups:
o First step uses oxaloacetate
o Last step produces oxaloacetate
o Utilization and regeneration of it is the cycle part
of the TCA cycle
Energy released in the oxidation reactions of TCA cycle
is conserved in form of reduced coenzymes that contain
a pair of electrons (NADH and FADH2)
These reduced electron carriers or reducing equivalents
are then transferred to the mitochondrion via shuttle

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes








mechanisms; oxidized by the electron transport chain


(found in the inner mitochondrial membrane) to
generate to ATP via the process of oxidative
phosphorylation, thus increasing energy yield of glucose
oxidation.
Hence, TCA produces the greatest amount of energy
(>25%) used by aerobic cells in conjunction with
oxidative phosphorylation
Oxidation,
o 1 NADH = 3 ATP
o 1 FADH2 = 2 ATP
Succinyl CoA thiokinase reaction generates 1 ATP at
substrate level phosphorylation
Hence, there are 12 ATPs generated from oxidation of 1
acetyl CoA per turn of cycle
Since there are 2 pyruvates entering mitochondrion and
counting starts from pyruvate, a total of 32 ATPs are
formed
TCA, thus defined as the final common pathway
because acetyl CoA (the common degradation product
of carbohydrate, lipid, and protein metabolism) is
oxidized to CO2 with ATP synthesis; most of the bodys
CO2 is produced by the decarboxylation reactions of the
cycle

CATABOLISM OF THE MAJOR NUTRIENTS CONVERGE


ON THE TCA CYCLE

ROLE OF VITAMINS IN TCA CYCLE


Table1. Essential B vitamins for the Krebs cycle
Vitamin

Active Form

Thiamine (B1)

Thiamine
pyrophosphate
(TPP)

Riboflavin (B2)

Flavin adenine
dinucleotide (FAD)

Niacin (B3)

Nicotinamide
adenine dinucleotide
(NAD)

Pantothenic acid

Component of
Coenzyme A

Function
Coenzyme for
decarboxylation in
the pyruvate
dehydrogenase
complex, isocitrate
dehydrogenase & ketoglutarate
dehydrogenase
complex
Cofactor of
succinate
dehydrogenase
Electron acceptor
for isocitrate
dehydrogenase, ketoglutarate
dehydrogenase
complex & malate
dehydrogenase
Cofactor attached to
active carboxylic
acid residues, such
as acetyl-coA and
succinyl coA

AMPHIBOLIC NATURE OF TCA CYCLE


Catabolic pathways
o Acetyl groups oxidized to form CO2 and energy is
conserved in the reduced coenzyme molecules
NADH and FADH2
 Anabolic pathways
o Several TCA intermediates are precursors in a
variety of biosynthetic pathways in different cell
types
*TCA does not operate in isolation


Carbohydrate

catabolism: Oxidation of Carbohydrates via


Glycolysis yields pyruvate; Pyruvate is converted to Acetyl
CoA in the presence of O2

1.

GLUCONEOGENESIS: all major members of the cycle


are potentially glycogenic (from citrate to oxaloacetate);
hence, they can be substrates for hepatic or renal
glucose synthesis via malate in times of starvation
Eg. Oxaloacetate converted to PEP via pyruvate
carboxylase and PEP carboxykinase

2.

AMINO ACID SYNTHESIS: via transamination reaction


a. Pyruvate is transaminated to alanine and other
amino acids
b. -ketoglutarate:

Transmitted to glutamate  converted to


other amino acids or purines

Converted to the neurotransmitter aminobutyric acid (GABA)


c. Oxaloacetate is transaminated to aspartate 
converted to other amino acids like asparagine;
urea, purines and pyrimidines (cytosine, uracil and
thymine)

3.

NITROGEN
METABOLISM:
aspartate
from
oxaloacetate serves as a source of nitrogen in the
production of arginosuccinate and purines; fumarate is
the end product which then enters the TCA cycle

Protein catabolism: Protein broken down to constituent


amino acids (carbon skeleton of these amino acids can
become a source of energy by being converted to acetyl
CoA when then enters the TCA cycle)
Fat metabolism: Fats hydrolyzed to fatty acids and
glycerol; glycerol is used as a substrate for hepatic
gluconeogenesis while fatty acids are converted to Acetyl
CoA via -oxidation or fatty acid spiral reactions
*Acetyl
CoA:
common
degradation
product
of
Carbohydrates, Proteins, and Fats.
*These major nutrients can be converted into energy
through TCA cycle and oxidative phosphorylation pathway
via acetyl CoA
*TCA is the most important link to the ETC where most of
the ATP needed for energy reactions is produced

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[Biochem]

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


4.

FATTY ACID AND STEROID SYNTHESIS:


a. In the well fed state, citrate can be removed from
the
cycle
to
serve
as
precursor
of
extramitochondrial acetyl CoA for fatty acid and
steroid synthesis
b. Citrate, transported from the mitochondrion into the
cytosol, is:
 split into acetyl CoA and oxaloacetate;
 reduced to malate via cytoplasmic malate
dehydrogenase;
 converted to malate via malic enzyme;
 converted to pyruvate and NADH2, a source of
reducing equivalents for biosynthetic processes
like fatty acid and cholesterol in the cytosol

5.

HEME SYNTHESIS:
a. Succinyl CoA can be removed from the cycle,
condense with glycine and serve as a precursor of
the porphyrins, biosynthesis of hemes in the liver
and bone marrow, cytochromes and other heme
proteins
b. Succinyl CoA may also be converted to succinate,
a Krebs Cycle intermediate
ANAPLEROTIC REACTIONS

Reactions wherein TCA intermediates are replenished


by special enzymatic reactions when these become
deficient
 Hence, anabolic processes drain the TCA of the
molecules to sustain its role in energy generation.
 In most of these anaplerotic reactions, amino acid
degradation forms the TCA intermediates:
a. Pyruvate carboxylase reaction (or the carboxylation
of pyruvate to oxaloacetate)
o The major anaplerotic reaction in the body. (ratelimiting step of gluconeogenesis).
o Pyruvate carboxylase catalyzes the transfer of CO2
(attached to biotin) into oxaloacetate in the
presence of ATP as energy donor.
o Deficient Oxaloacetate = pyruvate carboxylase
reactions proceed forward
o Excessive Oxaloacetate = reverse direction to
dispose pyruvate via TCA cycle
o The amino acids alanine and serine can enter the
TCA cycle via oxaloacetate and pyruvate upon
oxidation.
b. Glutamate is reversibly converted to -ketoglutarate
by transaminase (TA) and glutamate dehydrogenase
(GDH); glutamate may come from other amino acids;
this is the second major anaplerotic reaction in the
body.
c. Synthesis of succinyl CoA from odd chain fatty acids
and from isoleucine and valine via propionyl CoA.
d. Other amino acids can be degraded to fumarate.
e. Aspartate, alanine and serine can be degraded to
oxaloacetate.


c. Isocitrate dehydrogenase
d. -ketoglutarate dehydrogenase
2. Allosteric control is the main regulatory mechanism.
3. The activity of these enzymes is immediately dependent
on the supply of oxidized dehydrogenase cofactors like
NAD, ADP and ATP and dependent on the energy
status of the cell  expressed as [ATP]/[ADP] and
+
[NAHD]/[NAD ] ratio
A. Pyruvate Dehydrogenase
Allosteric inhibitors: ATP, NADH and acetyl CoA or in the
presence of sufficient energy supply of the cell as in a
+
resting state (or [ATP]/[ADP], [NAHD]/[NAD ] and [Acetyl
CoA]/[CoA] ratio.
 Acetyl CoA here exerts a product inhibition; it is,
+2
however, activated by Allosteric activator: Ca .
B. Citrate Synthase
Allosteric inhibitors: ATP, NADH, Succinyl CoA, longchain fatty acyl CoA, & citrate (a product inhibitor).
C. Isocitrate Dehydrogenase
Allosteric inhibitors: ATP & NADH
+
+2
Allosteric activators: ADP, NAD & Ca
+2
from contracting heart and other muscle
 Ca
tissues released from the ER to provide an
additional activator when ATP is being hydrolysed).
D. -Ketoglutarate Dehydrogenase
Allosteric inhibitors: by ATP, GTP, NADH & succinyl CoA
 succinyl CoA exerts a product inhibition or a
product regulates its own synthesis, hence ketoglutarate is available for biosynthetic reactions.
+2
Allosteric activator: Ca .
E.

CLINICAL CORRELATION


Control of TCA cycle is exerted at the level of the 4


enzymes that catalyze the non-equilibrium or
irreversible reactions, namely:
a. Pyruvate dehydrogenase
b. Citrate synthase
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[Biochem]

Beri-beri: a neurologic and cardiovascular disorder


caused by a dietary deficiency of thiamine (or Vitamin
B1 )
o common among the people of the Far East
because rice, the major food, has a rather low
content of thiamine.
o the problem is exacerbated if the rice is
polished because the outer layer contains
significant amounts of thiamine
o problem could be partly ameliorated if the
whole rice rain is soaked in water before
milling some of the thiamine in the husk then
leaves into the rice kernel.
Biochemical Basis of Beri-Beri

REGULATION OF KREBS CYCLE


1.

Malate dehydrogenase also allosterically inhibited by


ATP & NADH.

Thiamine pyrophosphate (TPP) is the prosthetic group


of 3 important enzymes:
pyruvate dehydrogenase
-ketoglutarate dehydrogenase
pyruvate carboxylase
TPP is deficient in beri-beri, hence pyruvate and ketoglutarate levels in the blood are elevated.
o Neurologic deficits are due to deficiency of
ATP to be used by the brain
o Leads to weakness of the musculature,
pain in the limbs, cardiac enlargement,
cardiac output and edema secondary to
decreased venous return

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


ATP YIELDS

SUMMARY OF ALL REACTIONS OF TCA CYCLE

ATP Yield per Molecule of Acetyl CoA Oxidized Through


the TCA Cycle

REACTANTS:
+
Acetyl CoA + 3 NAD + 1 GDP + Pi + 2 H2O

Table 2. ATP yield per molecule of acetyl CoA in TCA.

REACTION
MECHANISM
ATP YIELD
Isocitrate
NADH, OxPhos
3
dehydrogenase
-ketoglutarate
NADH, OxPhos
3
dehydrogenase
Succinyl CoA
Substrate-level
1
synthase
Phospho.
Succinate
FADH2, OxPhos
2
dehydrogenase
Malate
NADH, OxPhos
3
dehydrogenase
TOTAL
12
PYRUVATE  NADH, Oxid. Phospho. 
3
*2 pyruvates (glycolysis)  oxidized in TCA = 30ATPs
**Oxid. = oxidation; Phospho. = phosphorylation

PRODUCTS:
+
CoA-SH + 3 NADH + 3 H + 1 GTP + 2 CO2

II. PYRUVATE DEHYDROGENASE COMPLEX


GENERAL NATURE OF PYRUVATE DEHYDROGENASE
COMPLEX
One of the biggest enzymes (100 daltons)
Composed of:
o 3 enzymes (E1, E2, E3)
o 5 cofactors:

Catalytic cofactors (bound to the complex
subunits)

Thiamine pyrophosphate (TPP)

Lipoamide

FAD

Stoichiometric cofactors

CoA

NAD+

ATP Yield for Complete Oxidation of Glucose


Table 3. ATP yield for complete oxidation of Glucose
Mole ATP/
REACTION
MECHANISM
Mole
Glucose
A.Glycolysis
Hexokinase-Glucokinase Phospho.
-1
PFK-1
Phospho.
-1
G-3-PDH
NADH, Oxid., P
+6 (4)
Phosphoglycerate kinase Substrate-level P
+2
Pyruvate kinase
Substrate-level P
+2
B.TCA
Pyruvate dehydrogenase NADH, Oxid., P
+6
Isocitrate
NADH, Oxid., P
+6
dehydrogenase
NADH, Oxid., P
+6
-ketoglutarate
dehydrogenase
Substrate-level P
+2
+4
Succinyl CoA synthetase FADH2, Oxid., P
Succinate
dehydrogenase
NADH, Oxid., P
+6
Malate dehydrogenase
TOTAL
36-38 ATP
*Phospho/P = phosphorylation; Oxid. = oxidation
**1 NADH = 3 ATPs, 1 FADH = 2 ATPs

Note: Catalytic means they are used and are regenerated.


Stoichiometric means they are used, but not regenerated.
ENZYME

Abbr

Prosthetic
groups

Pyruvate
Decaboxylase

E1

TPP

Dihydrolipoyltransacetylase

E2

Lipoamide

Dihydrolipoyl
dehydrogenase

E3

FAD

Action
Oxidative
decarboxylation
of pyruvate
Transfer of
acetyl group to
CoA
Regenerates
oxidized
lipoamide

FATE OF PYRUVATE
A.

Pyruvate is transferred from:


Cytosol  Matrix of Mitochondria
Enzyme: Pyruvate Translocase

SHUTTLE MECHANISMS FOR CYTOPLASMIC


REDUCING EQUIVALENTS
1.

2.

3.

The cytoplasmic reducing equivalents (NADH from the


glycolytic glyceraldehyde 3-phosphate dehydrogenase
reaction step) are also sources of energy upon
+
reoxidation to NAD in the electron transport chain.
However, they are impermeable to the mitochondrial
membrane, hence their need for shuttle mechanisms
that transport them from the cytoplasm to the
mitochondrion.
The ATP yield therefore depends on the route of
transport of redox equivalents to the mitochondria:
a. Malate-Apartate Shuttle
= 6 ATPs
b. Glycerol Phosphate Shuttle = 4 ATPs

B.

Enzyme: Pyruvate Dehydrogenase Complex


Note:

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[Biochem]

Pyruvate is converted to acetyl CoA

th

 This is not the first step in TCA (Devlin, 4 ed)


 This is an irreversible and exergonic reaction

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


Step 5: Enzyme: Dihydroxylipoyl dehydrogenase (E3)

E3-FADH2 reacts with NAD+


+
Products: NADH + H + E3-FAD

PDH COMPLEX MECHANISM OF ACTION


(Refer to PPT for the structures of E1, E2 and E3)
Step 1: Enzyme: Pyruvate Decarboxylase (E1)

Pyruvate reacts with TPP of E1
Products:
CO2
+
HETPP
(hydroxyethylthiamine pyrophosphate)

PYRUVATE DEHYDROGENASE REGULATION

Step 2: Enzyme: Dihydroxylipoyltransacetylase (E2)



HETPP reacts with lipoamide of E2

2H from HETTP is transferred to lipoamide
Product: Dihydroxylipoamide + Acetyl-TPP

Acetyl component from Acetyl-TPP is transferred to
dihydroxylipoamide
Product: Acetyl-dihydroxylipoamide

The key means of regulation in eukaryotes is covalent


modification (phosphorylation or dephosphorylation) of
the E1 component of PDH complex
o PDH phosphatise dephosphorylates (activates)
2+
2+
PDH with Mg and Ca
o Mg2+-ATP dependent PDH kinase phosphorylates
(inactivates) PDH

A high ratio of NADH/NAD+, acetyl CoA/CoA
and ATP/ADP  Activates kinase 
Phosphorylation  Inactivation of PDH

More NAD+, CoA, pyruvate and ADP
(signalling low energy charge)  Inactivates
kinase  Promotes dephosphorylation by
phosphatase  Activation of PDH
In general, pyruvate dehydrogenase is switched off
when:
1. the energy charge is high; and,
2. biosynthetic intermediates are abundant.
(To prevent wastage)
ALTERATIONS IN PDH COMPLEX

A.

PDH Deficiency: Congenital Lactic Acidosis


Pyruvate is reduced to lactate instead of Acetyl CoA
E1 defect, X-linked dominant trait affects both male
and female
Neurodegeneration, muscle spasticity, death

B.
C.

Arsenic inactivates lipoamide arm of E2


Leigh Syndrome
Subacute necrotizing encephalomyelopathy
Defect in mitochondrial ATP production
complex, ETC, ATP synthase)
Nuclear and mtDNA affected

Step 3: Enzyme: Dihydroxylipoyltransacetylase (E2)

Acetyl group from Acetyl-dihyroxylipoamide is


transferred to CoA SH
Products: Acetyl CoA + Dihydroxylipoamide
Step 4: Enzyme: Dihydroxylipoyl dehydrogenase (E3)

Dihydroxylipoamide reacts with FAD of E3


Products: E2-Lipoamide + E3-FADH2

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[Biochem]

(PDH

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


Step 1:

III. PENTOSE PHOSPHATE PATHWAY


OVERVIEW OF PENTOSE PHOSPHATE PATHWAY

a.k.a. Hexose Monophosphate Shunt, WarburgDickens Pathway or Phosphogluconate Pathway

occurs in the cytosol







A. Two Phases of Pentose Phosphate Pathway


.
I. Oxidative Phase irreversible

Products: 2NADPH + CO2 + ribulose-5-PO4

Ribulose-5-PO4: for nucleotide biosynthesis

NADPH: for reductive pathways


e.g. fatty acid synthesis, drug detox, glutathione
defense system against injury by reactive oxygen
species

Step 2:






Substrate: Glucose-6-PO4 (from the glycolytic


pathway)
Enzyme: glucose-6-PO4 dehydrogenase
o once glucose enters the cell, it will be
phosphorylated to glc-6-PO4; another
pathway that glucose goes into in a wellfed state
Products: 6-phosphogluconolactone + NADPH
enough production of NADPH would inhibit the
activity of glc-6-PO4 dehydrogenase, this serves as
the rate limiting step
Substrate: 6-phosphogluconolactone
Enzyme: 6- phosphogluconolactonase
Product: 6-phosphogluconate
Dehydrogenase reaction with presence of Mg, Ca
and Mn
Gluconolactone formed from step 1 is rapidly
hydrolyzed to 6-phosphogluconate, with a
carboxylic acid group at C1. The next oxidation
step releases this carboxyl group as CO2, with the
electrons being transferred to NADP.

Step 3:





Overview of the Oxidative Phase of HMP(note the products
formed: NADPH & ribulose-5-PO4)



II. Non-oxidative Phase reversible

Products: fructose-6-PO4 or erythrose-4-PO4

Involves ribose precursors and intermediates that


can go back to glycolysis

2 enzymes: Transketolase and Transaldolase

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[Biochem]

Substrate: 6-phosphogluconate
Enzyme: 6-phosphogluconate dehydrogenase
Products:
Ribulose-5-PO4
(ketopentose)+
2NADPH + CO2
Form a 6-carbon intermediate, will then become a
5-carbon intermediate
Carbonyl carbon at C2

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


Step 6:

Step 4:








Substrate: Ribulose-5-PO4
Enzymes: Phosphopentoisomerase
Phosphopentoepimerase
Products: Ribose-5-PO4(aldopentose,
Xylulose-5-PO4(ketopentose)
The isomerase enzyme converts ribulose-5-PO4 to
ribose-5-PO4 while the epimerase enzyme
converts ribulose-5-PO4 to xylulose-5-PO4
Epimerase changes the stereochemical position of
one -OH group at carbon 3

Step 5:

Substrates: Ribose-5-PO4, Xylulose-5-PO4
st

Enzyme:Transketolase (1 )

Products:Sedoheptulose-7-PO4(7 carbon sugar),
Glyceraldehyde-3-PO4

Coenzymes of Transketolase:
1. Thiamine Pyrophosphate/TPP
- important for prolonging RBC lifespan
2. Mg 2+

Transketolase picks up a 2-C fragment from
xylulose-5-PO4 by cleaving the C-C bond between
the keto group and the adjacent carbon thus
releasing glyceraldehyde-3-PO4

The 2-C fragment is covalently bound to thiamine
pyrophosphate, which transfers it to the aldehyde
carbon of another sugar, forming a new ketose





Substrates:Sedoheptulose-7-PO4,
Glyceraldehyde-3-PO4
Enzyme:Transaldolase
Products:Erythrose-4-PO4(4-C sugar)
Fructose-6-PO4(6-C sugar)
Fructose-6-PO4 can be converted to glucose-6PO4 so it serve as an intermediate of the same
pathway; it can also go back to the glycolytic
pathway
The aldol cleavage occurs between the two OH
carbons adjacent to the keto group(on carbons 3
and 4 of sugar)

Step 7:

Substrates: Xylulose-5-PO4, Erythrose-4-PO4
nd

Enzyme: Transketolase (2 )

Products: Glyceraldehyde-3-PO4
Fructose-6-PO4

This step serves as a CONTROL, wherein there is
reversal by the transaldolase and transketolase
enzymes

If
body
needs
more
ribose-5-PO4,
the
transketolase enzyme is the one activated

If body needs more glucose (undergo glycolysis
and gluconeogenesis), the transaldolase enzyme
is the one activated
B. Differences between Glycolysis and Pentose Phosphate
Pathway
Table 4. Table Comparing Glycolysis and PPP
PENTOSE
FEATURES
GLYCOLYSIS
PHOSPHATE
Substrate and
G-6-PO4
Substrate
end product
CO2
Not a product
Product
Hydrogen
+
+
NAD
NADP
Acceptor
ATP Generation
Major function
None
Ribose-5-PO4
None
Present
Generation

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[Biochem]

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


C. Biomedical Importance of Pentose Phosphate
Pathway
1.

IV. URONIC ACID PATHWAY


URONIC ACID PATHWAY OVERVIEW

Prolongs RBC lifespan


Oxidized Glutathione
+
Glutathione + NAD

NADPH

------

Reduced

Enzyme: Glutathione Reductase


Reduced Glutathione + H2O2 ------- Oxidized Glutathione
+ H2O

catalyzes the conversion of glucose to glucuronic acid,


ascorbic acid, and pentoses
an alternative oxidative pathway for glucose metabolism
does not lead to the formation of ATP

STEP 1: Glucose 6-phosphate is converted to Glucose


1-phosphate via phosphoglucomutase

Enzyme: Glutathione Peroxidase


2.

HMP in RBC provides NADPH for reduction of


oxidized glutathione in the presence of glutathione
reductase
In turn, reduced glutathione removes H2O2 in RBC,
catalyzed by glutathione peroxidase.
Hence H2O2 is prevented from oxidizing Hgb to
methemoglobin
increased RBC lifespan no RBC hemolysis

Hemolytic Anemia

In RBC, Glucose-6-PO4 Dehydrogenase is an


important enzyme for mantaining membrane
integrity of RBC.
Glucose-6-PO4 Dehydrogenase catalyzes the first
step in PPP/HMP which produces NADPH.
In glucose-6-PO4 Dehydrogenase deficiency, there
is deficient NADPH.
Therefore, there is defective glutathione peroxidase
activity, leading to inhibition of H2O2 detoxification.
o H2O2 is not prevented from oxidizing
Hgb to methemoglobin.
breakdown of RBC membranes  Hemolytic
Anemia
Note: Drugs that can cause Glucose-6-PO4 Dehydrogenase
deficiency.
Antimalarial Drugs (primaquine)
Chloramphenicol
Aspirin
Sulfonamide
Foods: Fava beans
D. Other pathways that require NADPH
1. REDUCTIVE SYNTHESIS (ANABOLIC PATHWAYS)
a. Fatty acid synthesis
b. Fatty acid chain elongation
c. Cholesterol synthesis
d. Neurotransmitter synthesis
e. Nucleotide synthesis
f. Superoxide synthesis
2. DETOXIFICATION
a. Cytochrome P450 monooxygenase NADPH is the
source of reducing equivalents for cytochrome P450
hydroxylation of aromatic compounds, steroids,
alcohol and drugs detoxification.
b. NADPH maintains levels of reduced glutathione
important for:
*RBC membrane integrity
*Defense of the body against injury by reactive oxygen
species (ROS)
-

STEP 2: Glucose 1-phosphate reacts with uridine


triphosphate (UTP) via UDP glucosepyrophosphorylase
to form UDP glucose.

STEP 3: UDP glucose is oxidized at C6 by a 2-step


+
process via an NAD -dependent UDP glucose
dehydrogenase to form UDP glucuronic acid

UDP glucuronic acid = activated glucuronic acid

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[Biochem]

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


UDP-glucuronate

source of glucuronate for reactions involving its


incorporation into proteoglycans

conjugated to nonpolar acceptor molecules such as


steroid hormones, some drugs, bilirubin, or other
foreign compounds in the liver for easier excretion
via the bile

STEP 7: Decarboxylation of 3-Keto-L-Gulonic Acid

STEP 4: UDP glucuronic acid is hydrolysed to form UDP


and D-glucuronic acid

STEP 5: Oxidation of D-glucuronic acid to L-gulonic acid


via L-gulonic dehydrogenase in the presence of
NADPH2.

L-gulonate

the direct precursor of ascorbate in those animals


capable of synthesizing this vitamin, in an NADPHdependent reaction
o In humans, ascorbic acid cannot be
synthesized because of the absence of Lgulonolactone oxidase

STEP 8: Oxidation of L-Xylulose

L-xylulose is then reduced to xylitol via xylitol


dehydrogenase (or xylulosereductase)

STEP 9: Reoxidation of Xylitol

STEP 6: Oxidation of L-Udonic acid

L-gulonic acid may be oxidized to 3-keto-L-gulonic


acid via -L-hydroxy acid dehydrogenase; NADH is
generated.
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Followed by decarboxylation of 3-keto-L-gulonic


acid to form L-xylulose,
a ketopentose via -Lgulonate decarboxylase; here, carbon1 of 3-keto-Lgulonic acid is released as CO2.

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


STEP 10: Phosphorylation of D-Xylulose

D-xylulose is phosphorylated at carbon 5 to form Dxylulose 5-phosphate via xylulose kinase further
metabolized via the HMP Shunt  converted to
intermediates of glycolysis for energy production.
In essential pentosuria, xylitol dehydrogenase is
absent:
L-xylulose cannot be reduced to xylitol 
accumulation of L-xylulosein the blood  large
amounts of L-xylulose are seen in the urine of
patients, especially following intake of glucuroniccid.
rare, non-fatal genetic disease.

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[Biochem]

[BIOCHEM] 2.4 TCA/PDH/HPM/Uronic Acid Pathways Dr. Reyes


III. Pentose Phosphate Pathway (or HMP)

APPENDIX
I.

Tricarboxylic Acid Pathway

IV. Uronic Acid Pathway

II.

Pyruvate Dehydrogenase Complex

CoASH

CoA

NAD+
NADH

Source: http://www3.nd.edu/~aseriann/pyrde.html

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