Download Redox Reactions in Metabolism Supplemental Reading Key

Bioc 460 - Dr. Miesfeld Spring 2008
Redox Reactions in Metabolism Supplemental Reading
Key Concepts
- Reduction potentials are a measurement of electron affinity
- Coenzymes provide reactive groups that function in enzyme catalysis
- The pyruvate dehydrogenase complex is a metabolic machine
KEY CONCEPT QUESTIONS IN METABOLIC REDOX REACTIONS:
What does the ΔE value of a coupled redox reaction tell you about electron transfer potential?
What are coenzymes and how do they function in the pyruvate dehydrogenase reaction?
Biochemical Applications of Coenzyme Biochemistry:
Thiamin, also called vitamin B1, is an important
enzyme cofactor required for a variety of metabolic
reactions. Beriberi is a disease caused by thiamin
deficiency resulting in severe weight loss and
neurological symptoms. People that eat polished white
rice as a sole source of nourishment can develop
beriberi because polished rice lacks thiamin. Foods
rich in thiamin include watermelon, sunflower seeds,
black beans and thiamin enriched grains and breads.
Reduction potentials are a measurement of electron affinity
Before we begin discussing the Citrate Cycle (lecture 28), we need to describe several biological
redox reactions (oxidation-reduction) that represent a form of energy conversion involving the
transfer of electron pairs from organic substrates to the carrier molecules NAD+ and FAD. The
energy available from redox reactions is due to differences in the electron affinity of two
compounds and is an inherent property of each molecule based on molecular structure. Since
electrons do not exist free in solution, electrons must be passed from one compound to another in
a coupled redox reaction. Coupled redox reactions consist of two half reactions, 1) an oxidation
reaction (loss of electrons) and 2) a reduction reaction (gain of electrons). Compounds that
accept electrons are called oxidants and are reduced in the reaction, whereas compounds that
donate electrons are called reductants and are said to be oxidized by loss of electrons.
Redox reactions in biochemistry rarely involve molecular oxygen (O2) directly, but rather are
characterized by the loss and gain of electrons from carbon. The terminology of biochemical
redox reactions is the same as that used in inorganic chemistry. Namely, each half reaction
consists of a conjugate redox pair represented by a molecule with and without an electron (e-).
For example, Fe2+/Fe3+ is a conjugate redox pair in which the ferrous ion (Fe2+) is the reductant
that loses an e- during oxidation to become a ferric ion (Fe3+):
Fe2+ <--> Fe3+ + ereductant
oxidant
Similarly, the reductant cuprous ion (Cu+) can be oxidized to form the oxidant cupric ion (Cu2+) plus
an e- in the reaction:
Cu+ <--> Cu2+ + ereductant
oxidant
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The two conjugate redox pairs in these reactions are Fe2+/Fe3+ and Cu+/Cu2+. We can now
combine these two half reactions into a coupled redox reaction by reversing the direction of the
Cu2+ reduction reaction such that the e- functions as the "common intermediate" shared by the
Fe2+ oxidation and Cu2+ reduction half-reactions:
Fe2+ <--> Fe3+ + e- (oxidation of Fe2+)
Cu2+ + e- <--> Cu+ (reduction of Cu2+)
Fe2+ + Cu2+ <--> Fe3+ + Cu+ (coupled redox reaction)
The oxidation of Fe2+ and reduction of Cu2+ is a coupled redox reaction we will see in lecture 29
when we examine the function of the cytochrome c oxidase complex in the electron transport
system.
Figure 1.
The combination of glycolysis, the citrate cycle and the
electron transport system result in the complete
oxidation of glucose to form CO2 and H2O by a process
called aerobic respiration. The e- donor is glucose
which functions as the reductant, and O2 is the eacceptor (oxidant) that is reduced in the last step of the
electron transport system to form H2O. The two
conjugate redox pairs NAD+/NADH and FAD/FADH2
serve as the e- carriers linking glycolysis to the citrate
cycle and electron transport chain. It is useful to think
of glucose as biochemical "battery" containing
stored energy in the form of electrons that can be used
to synthesize ATP in the mitochondria as a result of
proton motive force and oxidative phosphorylation.
Figure 1 illustrates the relationship between methane
(CH4), the most highly reduced form of carbon which
has 8 e- that can be donated, and carbon dioxide
(CO2) in which all of the e- shared by the C and O are
tightly associated with the more electronegative O. The
more electrons a carbon atom has available to donate in a redox reaction, the more reduced (less
oxidized) it is. Hydrogen is less electronegative than carbon, and therefore electrons in C-H bonds
are considered "owned" by the carbon. Similarly, since oxygen is more electronegative than
carbon, the electrons in C-O and C=O bonds are all "owned" by the oxygen atom. Note that in
biological redox reactions, often (but not always), an increase in oxidation state of a carbon is
associated with a decrease in the number of hydrogen atoms.
Unlike the oxidation of Fe3+ which simply involves the transfer of one e- to Cu2+, redox reactions in
the citrate cycle (and indeed most all enzyme-catalyzed redox reactions) involve the transfer of
electron pairs (2 e-) to the electron carrier molecules NAD+ and FAD. The reduction of NAD+ to
NADH involves the transfer of a hydride ion (:H-), which contains 2 e- and 1 H+, and the release of
a proton (H+) into solution
NAD+ + 2 e- + 2 H+ <--> NADH + H+
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In contrast, FAD is reduced by sequential addition of one hydrogen (1 e- and 1 H+) at a time to give
the fully reduced FADH2 product
FAD + 1 e- + 1 H+ <--> FADH + 1 e- + H+ <--> FADH2
Oxidations can also involve a direct combination with oxygen which oxidizes the carbon by pulling
e- toward the more electronegative O atom. Enzymes that catalyze biochemical redox reactions
are strictly called oxidoreductases, however, since most oxidation reactions involve the loss of
one or more hydrogen atoms, they are often called dehydrogenases. We will look at the
reduction of the coenzymes NAD+ and FAD by dehydrogenases in more detail later.
The two primary energy conversion reactions in metabolism are 1) phosphoryl transfers
involving ATP, and 2) redox reactions that transfer pairs of electrons between organic compounds
and the electron carriers NAD+/NADH and FAD/FADH2. As we discussed in lecture 24, the change
in standard free energy of a reaction under biochemical conditions (ΔGº') is a measure of the
spontaneity of the reaction in kJ/mol and reflects the tendency of compound A to be converted to
compound B (A --> B). A negative ΔGº' (ΔGº' < 0) means the reaction is favored in the direction
written from left to right (product B will accumulate), whereas, a positive ΔGº' (ΔGº' > 0) means the
reverse reaction is favorable (A will accumulate).
Figure 2.
In redox reactions, we use the term reduction potential
(E), measured in volts (V), to represent the electron affinity
of a given conjugate redox pair. Analogous to biochemical
standard conditions that define Gibbs Free Energy, Gº',
(25ºC, pH 7 and 1 M initial concentration of substrates and
products), the term Eº' refers to the biochemical
standard reduction potential under the same conditions.
Figure 2 illustrates how Eº' values are determined in the
laboratory using an apparatus called an electrochemical
cell that measures the relative e- affinity of a test redox
pair, compared to that of the hydrogen half-reaction (2H+
+ 2e- <--> H2), which has been chosen as the standard.
For these measurements, two half-cells are connected by
a type of voltmeter (galvanometer) and platinum
electrodes that measure the movement of electrons from
one half cell to the other. Depending on the relative
electron affinity of the test oxidant compared to H+, the
electrons will either move from the hydrogen half-cell toward the test half-cell, or from the test
half-cell toward the hydrogen half-cell. The two half-cells are connected by an agar bridge
containing potassium chloride that permits counter-ion movement to balance the charge. The Eº' of
oxidants with a higher affinity for electrons than H+ are recorded as positive Eº' values (Eº' > 0),
and oxidants with a lower affinity for electrons than H+ are have negative Eº' values (Eº' < 0). In
the example shown in figure 2, under standard conditions of 25ºC and 1 atmosphere of pressure,
the hydrogen half cell on the right is assigned the arbitrary Eº' value of 0.00. It can be seen that
the voltmeter registers a Eº' value of +0.771 volts, meaning that the electrons flow from the
hydrogen reference cell toward the Fe3+/Fe2+ test cell demonstrating that Fe3+ has a higher affinity
for electrons than H+.
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Figure 3 lists the Eº' values that
have been measured for a
number of conjugate redox pairs
in biochemical reactions. It can
be seen that O2 is the most
potent oxidant in the table with a
Eº' value of +0.816 and will
therefore readily accept
electrons from all other
conjugate redox pairs shown,
whereas, α-ketoglutarate is a
very weak oxidant (strongest
reductant) and will donate
electrons to other conjugate
redox pairs in the table. By
convention, standard reduction
potentials are expressed as halfreactions written in the direction
of a reduction reaction.
Therefore, when a oxidation
reaction is written in the reverse
direction, the sign of the Eº'
value needs to change
accordingly (e.g., from + to -).
The table highlights some of the
redox reactions we will
encounter in metabolism.
Figure 3.
The amount of energy available
from a coupled redox reaction is
directly related to the difference between two reduction potentials and is defined by the term ΔEº'.
By convention, the ΔEº' of a coupled redox reaction is determined by subtracting the Eº' of the
oxidant (e- acceptor) from the Eº' of the reductant (e- donor) using the following equation:
ΔEº' = (Eº'e- acceptor) - (Eº'e- donor)
Moreover, the ΔEº' for a coupled redox reaction is proportional to the change in free energy
ΔGº' as described by the equation:
ΔGº' = -nFΔEº'
in which n is the number of electrons transferred in the reaction (usually 2 in biochemical redox
reactions), and F is the Faraday constant (96.48 kJ/V•mol). As can be seen by this equation, when
the difference in reduction potentials for a coupled redox reaction is positive (ΔEº' > 0) then the
reaction is favorable since ΔGº' will be negative. Looking back at the definition of ΔEº', this
means that for a coupled redox reaction to be favorable, the reduction potential of the eacceptor needs to be more positive than that of the e- donor.
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To see how ΔGº and ΔEº are related, we can use the biochemical standard reduction potentials
(Eº') in figure 3 to calculate the change in biochemical standard free energy (ΔGº') for the citrate
cycle isocitrate dehydrogenase reaction. Note that in a spontaneous coupled redox reaction the
e- flow is from the reductant in the conjugate redox pair with the lower Eº' value (more negative)
toward the oxidant in the conjugate redox pair with the higher Eº' value (less negative). The two
standard half reactions from figure 3 are written below as reduction reactions. Note that NAD+ is
the e- acceptor (less negative) and α-ketoglutarate is the e- donor (more negative):
α-ketoglutarate + CO2 + 2 e- + 2 H+ --> Isocitrate
(Eº' = -0.38 V)
NAD+ 2 e- + 2 H+ --> NADH + H+
(Eº' = -0.32 V)
We can calculate ΔEº' for this coupled redox reaction using the equation below:
ΔEº' = (Eº'e- acceptor) - (Eº'e- donor)
ΔEº' = (Eº'NAD+) - (Eº'isocitrate)
ΔEº' = (-0.32 V) - (-0.38 V) = +0.06 V
and then convert this ΔEº' value to ΔGº' using the relationship:
ΔGº' = -nFΔEº'
ΔGº' = -2 • (96.48 kJ/mol•V) • +0.06 V
ΔGº' = -11.6 kJ/mol
showing that the conversion of isocitrate to α-ketoglutarate is a favorable reaction (ΔGº' < 0) under
standard biochemical conditions.
Note that since biochemical conditions inside the mitochondrial matrix are not standard, in order to
calculate the actual reduction potentials for conjugate redox pairs, we need to take into account the
concentration of the oxidant (e- acceptor) and reductant (e- donor) using an equation described by
Walther Nernst in 1881:
E = Eº' + RT • ln [e- acceptor]
nF
[e- donor]
In the Nernst equation, R is the gas constant (8.314 J/Kº • mol), T is the absolute temperature in
Kelvin (Kº), n is number of electrons transferred and F is the Faraday constant (96.48 kJ/V•mol).
Coenzymes provide reactive groups that function in enzyme catalysis
Pyruvate is a three carbon metabolite derived from glucose or amino acids that must be
transported from the cytosol into the mitochondrial matrix before it can serve as a source of
reducing power for the cell (citrate cycle) or as a precursor for glucose synthesis
(gluconeogenesis). Pyruvate that is destined for the citrate cycle (or fatty acid synthesis) is
converted to acetyl CoA by the enzyme pyruvate dehydrogenase. As shown in figure 4, acetylCoA has only two metabolic fates in the cell, 1) it can be metabolized by the citrate cycle to
convert redox energy to ATP by oxidative phosphorylation, or 2) it can be used as a form of
stored energy by conversion to fatty acids that are transported to adipocytes (fat cells) as
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triglycerides. Since the pyruvate dehydrogenase reaction is irreversible (ΔGº’ = -33.4 kJ/mol),
production of acetyl-CoA by the pyruvate dehydrogenase reaction is tightly controlled to coordinate
energy needs of the cell with the production of acetyl-CoA.
Figure 4.
This is especially important in animals which lack the necessary
enzymes to convert fats to carbohydrates, and therefore,
cannot reutilized acetyl-CoA for glucose production when
carbohydrate levels are low. Because of this, the pyruvate
dehydrogenase complex is only fully active in animal cells when
carbohydrate sources are plentiful.
The pyruvate dehydrogenase complex catalyzes the
oxidative decarboxylation of pyruvate to form CO2 and acetylCoA using a five step reaction mechanism that requires three
distinct enzymes and five different coenzymes. We begin by
first looking at the important role of coenzymes in metabolic
reactions, specifically NAD+, FAD, CoA, thiamine
pyrophosphate (TPP) and lipoic acid which are all utilized in the
pyruvate dehydrogenase reaction.
Amino acids can only provide a finite number of chemical
groups for enzyme reactions, and moreover, structural
constraints of the polypeptide backbone restrict the precise
positioning (orientation) of amino acid side groups within the
active site. However, enzyme complexes that bind reactive
biomolecules through covalent or non-covalent interactions provide additional reactive groups for
catalytic mechanisms. These biomolecules are called enzyme cofactors or coenzymes and are
often complex organic compounds that are obtained as nutrients in the diet. The term vitamin
describes organic molecules that are required in the diet, but which do not contribute directly to
energy conversion through catabolism, or do not provide a structural role to other biomolecules.
Figure 5 lists six coenzymes in metabolism and their role in enzymatic reactions.
Figure 5.
Coenzyme
Nicotinamide adenine
+
dinucleotide (NAD )
Vitamin
Niacin (B3)
Types of reactions
Redox reactions
(transfer of hydride ion)
Nutrient source
Poultry, fish,
vegetables
Symptoms of dietary
deficiency
Causes the disease
pellagra
Flavin adenine
dinucleotide (FAD)
Riboflavin
(B2)
Redox reactions
(transfer of electrons)
Dairy, almonds,
asparagus
Causes cheilosis
(swelling,cracked lips)
Coenzyme A (CoA)
Pantothenic
acid (B5)
Acyl group transfer
Chicken, yogurt,
avocados
Rarely observed
Thiamin
pyrophosphate (TPP)
Thiamin (B1)
Aldehyde transfer
Lentils, brown rice,
fortified cereals
Causes beriberi
α-Lipoic acid
(lipoamide)
Not a
vitamin
Acyl group transfer
Tomatoes, broccoli,
spinach
None reported
Biocytin (biotin-lysine)
Biotin
Carboxyl group transfer
Breads, cooked
eggs, vegetables
skin rash, hair loss,
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Nicotinamide adenine dinucleotide (NAD+) - is derived from the water-soluble vitamin niacin
which is also called vitamin B3. NAD+, and its phosphorylated form NADP+, are involved in over
200 redox reactions in the cell which are characterized by the transfer of 2 e- in the form of hydride
ions (:H-). Catabolic redox reactions primarily use the conjugate redox pair NAD+/NADH and
anabolic reactions use NADP+/NADPH. The structure of the oxidized and reduced forms of
NAD(P)+ and NAD(P)H, respectively, are shown in figure 6. Note that the "+" charge does not
refer to the overall charge of the NAD molecule, but rather only to the charge on the ring N in the
oxidized state.
Figure 6.
Severe niacin deficiency causes the disease
pellagra which was first described in Europe
in the early 1700s amongst peasants who
relied on cultivated corn as their primary
source of nutrition. Although it was initially
thought that pellagra was caused by an
infectious agent in contaminated corn,
nutritional studies showed that it was due to
insufficient levels of bioavailable niacin in a
corn-rich diet. Interestingly, pellagra is rare in
Mexico because corn used for tortillas is
traditionally soaked in lime solution (calcium
oxide) prior to cooking and this releases
niacin from its bound form upon heating.
Flavin adenine dinucleotide
Figure 7.
(FAD) - is derived from the
water-soluble vitamin
riboflavin which is also
called vitamin B2. Riboflavin
is the precursor to FAD and
to the related molecule flavin
mononucleotide (FMN), both
of which are often tightly
associated with enzymes that
catalyze redox reactions.
FAD is a coenzyme in the
pyruvate dehydrogenase
complex and is also
covalently bound to a
histidine residue in the citrate
cycle enzyme succinate
dehydrogenase. FAD is
reduced to FADH2 by the
transfer of two electrons in
the form of hydrogen atoms (figure 7). Unlike NAD, FAD can accept one electron at a time and
form a partially reduced intermediate called a semiquinone (FADH•). Foods that have been found
to be high in riboflavin include dairy products (milk, cheese, eggs), almonds and asparagus.
Riboflavin, like several other vitamins, is destroyed by light which is one reason why milk is no
longer stored in clear containers.
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Coenzyme A (CoA) - is derived from the water-soluble vitamin pantothenic acid which is also
called vitamin B5. CoA is absolutely essential for life as it is required for energy conversion by the
citrate cycle, it is
Figure 8.
also a cofactor in
fatty acid,
acetylcholine,
heme and
cholesterol
biosynthetic
pathways. The
primary role of CoA
is to function as a
carrier molecule for
acetate units in
the form of acetyl-CoA. Figure 8 shows the structure of acetyl-CoA which consists of a central
pantothenic acid unit that is linked to a functional β-mercaptoethylamine group derived from
cysteine, and to adenosine 3,5-diphosphate. The acetate unit is covalently attached to CoA
through an activated thioester bond which has a high standard free energy of hydrolysis making it
an ideal acyl carrier compound. As such, attachment of acetate units to the reduced form of CoA
(CoA-SH) requires reactions with high ΔGº' values, for example, pyruvate dehydrogenase (ΔGº' = 33.4 kJ/mol) and α-ketoglutarate dehydrogenase (ΔGº' = -33.5 kJ/mol).
Thiamin pyrophosphate (TPP) - is derived from the Figure 9.
water-soluble vitamin thiamin (or thiamine) which is
also called vitamin B1. The structure of TPP is shown
in figure 9 where it can be seen that a carbon atom
on the thiazole ring is the functional component of
the coenzyme involved in aldehyde transfer. Thiamin
is absorbed in the gut and transported to tissues
where it is phosphorylated by the enzyme thiamin
kinase in the presence of ATP to form thiamin
pyrophosphate (TPP) and AMP.
Thiamin deficiency was first described in Chinese literature over four thousand years ago and is
the cause of beriberi, a disease characterized by anorexia, cardiovascular problems and
neurological symptoms. Beriberi has been found in populations that rely on white polished rice as
a primary source of nutrition (milling rice removes the bran which contains thiamin) and diets rich in
foods that contain the enzyme thiaminase which degrades thiamin during digestion. Raw fish
contains thiaminase, as does African silkworms a favorite food in some Nigerian cultures. Cooking
these foods destroys the thiaminase and alleviates the symptoms of beriberi.
α -Lipoic acid (lipoamide) - is a coenzyme synthesized in plants and animals as a 6,8dithiooctanoic acid. The role of α-lipoic acid in metabolic reactions is to provide a reactive disulfide
that can participate in redox reactions within the enzyme active site. Lipoamide, the naturally
occurring form of α-lipoic acid, is a covalent linkage of α-lipoic acid to a lysine ε-amino group on
proteins. The structure of lipoamide is shown in figure 10 where it can be seen that the long
hydrocarbon chain bridging -lipoic acid and lysine provides a flexible extension to the reactive
thiol group.
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As illustrated in figure 10, the E2 subunit of the
pyruvate dehydrogenase complex contains the
lipoamide at the end of a polypeptide tether
which functions as a "ball and chain" that
moves the lipoamide back and forth across a
50 Å span in the interior of the complex. αLipoic acid is not considered a vitamin
because it is synthesized at measurable levels
in humans, however, because of its potential
to function as an antioxidant in the reduced
form, α-lipoic acid is promoted as a nutritional
supplement. High levels of α-lipoic acid are in
broccoli, liver, spinach and tomato.
Figure 10.
The pyruvate dehydrogenase complex is a
metabolic machine
The conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase complex is an oxidative
decarboxylation reaction that represents another amazing example of protein structure and
function. The eukaryotic pyruvate dehydrogenase complex contains multiple subunits of three
different catalytic enzymes that work together as a metabolic machine to carry out the following net
reaction:
Pyruvate + CoA + NAD+ ---> acetyl-CoA + CO2 + NADH
ΔGº’ = -33.4 kJ/mol
Coenzymes perform a critical role in the pyruvate dehydrogenase complex by providing a chemical
platform for the
Figure 11.
catalytic
reactions. Three
of the coenzymes
are covalently
linked to enzyme
subunits, with
TPP attached to
the E1 pyruvate
dehydrogenase
subunit,
lipoamide is the
functional
component of the
E2 dihydrolipoyl
transacetylase
subunit, and FAD
is covalently
bound to the E3
dihydrolipoyl
dehydrogenase
subunit. The two
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other coenzymes, CoA and NAD+, are transiently associated with the E2 and E3 complexes,
respectively. The pyruvate dehydrogenase reaction can be broken down into five distinct catalytic
steps in which steps 1, 2 and 3 lead to the formation of acetyl-CoA, with steps 4 and 5 serving to
regenerate the oxidized from of lipoamide and in the process, transfer 2 e- to NAD+ (figure 11).
Step 1 - The E1 subunit (pyruvate dehydrogenase) binds pyruvate and catalyzes a decarboxylation
reaction resulting in the formation of hydroxyethyl-TPP and the subsequent release of CO2.
Step 2 - The hydroxyethyl-TPP of E1 reacts with the disulfide of the lipoamide group on the Nterminal domain of the E2 subunit (dihydrolipoyl transacetylase) to generate acetyldihydrolipoamide through a thioester bond.
Step 3 - The E2 lipoamide domain carries the acetyl group from the E1 catalytic site across a ~50
Å gap in the complex to the E2 catalytic site where it reacts with CoA-SH to yield acetyl-CoA and
fully reduced dihydrolipoamide.
Step 4 - The lipoamide domain then swings over to the E3
subunit (dihydrolipoyl dehydrogenase) where it is reoxidized to
the disulfide by a transfer of 2 e- and 2 H+ to a disulfide
contained on the E3 subunit; the dithiol is reoxidized by
transferring 2 e- and 2 H+ to the E3-linked FAD moiety to
transiently form E3-FADH2.
Figure 12.
Step 5 - The E3-FADH2 coenzyme intermediate is reoxidized in
a coupled redox reaction that transfers the 2 e- to NAD+ as a
hydride ion (:H-) leading to the formation of NADH + H+.
As shown in figure 12, the E1, E2 and E3 subunits of the
mammalian pyruvate dehydrogenase are packed together in a
huge ~400 Å diameter sphere with a combined molecular
weight of ~7800 kDa. The colored E1 (yellow),
Figure 13.
E2 (green) and E3 (red) pyruvate
dehydrogenase complex subunits are labeled,
and the linker region connecting E2 to E1 is
shaded gray. The stoichiometry of the E1:E2:3
subunits (22:60:6) is consistent with there
being 60 active sites in the pyruvate
dehydrogenase complex.
Figure 13 shows that the lipoamide moiety of
the E2 subunit is attached near the end of a
~200 amino acid long segment of the protein
that functions as both a structural linker
connecting the E2 and E1 subunits (gray
region in figure 16), and as a type of lipoamide
"ball and chain." An important component of
the linker region is the E1-binding domain that
serves as a "pivot" for the ball and chain. The
hydrocarbon extension on the lipoamide
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moiety itself is sometimes referred to as a "swinging arm." Because of the relative positioning of
E2 and E1 subunits with the pyruvate dehydrogenase complex, a single E2 subunit can "harvest"
hydroxyethyl groups from multiple E1 catalytic sites and deliver them to the E2 subunit.
The lipoamide group is the workhorse in this catalytic machine, and without a fully functional
pyruvate dehydrogenase complex, the link between glycolysis and the citrate cycle would be
broken. A naturally occurring inhibitor of lipoamide coenzyme function is the element arsenic (As)
which in the form of arsenite (AsO33-) creates bidentate adducts on dihydrolipoamide as shown in
figure 14. Inadvertent ingestion arsenite can lead to an untimely death by irreversibly blocking the
catalytic activity of lipoamide-containing enzymes such as the pyruvate dehydrogenase and αketoglutarate dehydrogenase complexes.
Figure 14.
Figure 15.
Chronic arsenic poisoning can come from environmental sources such as arsenic-contaminated
drinking water or household paints and results in the appearance of ulcerous skin lesions and an
increased risk of a variety of cancers. While stories abound that arsenic poisoning was used
routinely to kill off kings and queens in the Middle Ages, and may have even been involved in the
death of the exiled French general Napoleon Bonaparte, a more tragic example is that of
accidental arsenic poisoning of thousands of people in Bangladesh, India, which has occurred over
the last 20 years.
Since the 1990s it has been documented that millions of people in India have been
chronically exposed to toxic levels of arsenic in contaminated drinking water obtained from shallow
hand-pumped wells (figure 15). During the 1970s and 1980s, UNICEF and other relief
organizations helped drill thousands of wells in small Indian villages as an humanitarian effort to
circumvent public water supplies that had become biologically contaminated. About ten years later
when large numbers of villagers in the Ganges delta region developed skin lesions and cancers, it
was realized that these wells contained water with toxic levels of arsenic. Massive efforts were
undertaken to close down contaminated wells and to develop purification systems to reduce the
arsenic to safe levels in other water supplies. Arsenic-contaminated drinking water has also been
found in Southeast Asia and South America, usually near areas that had been extensively mined.
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ANSWERS TO KEY CONCEPT QUESTIONS IN THE CITRATE CYCLE:
The difference in reduction potential (ΔE) for a coupled redox reaction represents the tendency of
the reductant in one conjugate redox pair to donate electrons to the oxidant of the other conjugate
redox pair. Since the ΔE value of a redox reaction is proportional to the change in free energy
(ΔG) as described by the equation ΔG = -nFΔE, redox reactions with positive ΔE values (ΔG<0) are
more favorable than reactions with negative ΔE values (ΔG>0). The reduction potential for a given
conjugate redox pair is determined experimentally using an apparatus that measures electron flow
from one half-cell to another. Electrons move through platinum wires from the half cell containing a
reductant with low electron affinity toward the half-cell containing an oxidant with high electron
affinity. In many biochemical redox reactions, electrons are transferred in pairs (2 e-) from one
molecule to another, sometimes as hydride ions (:H-), as is the case with the coenzyme NAD+
(NAD+ + H+ + 2 e- <--> NADH).
Coenzymes are biomolecules that provide additional functional groups to enzyme active sites and
participate in catalytic mechanisms. The pyruvate dehydrogenase complex consists of multiple
copies of three protein subunits (E1, E2, E3) and requires five coenzymes (thiamin pyrophosphate;
TPP, lipoamide, coenzyme A; CoA, flavin adenine dinucleotide; FAD, and nicotinamide adenine
dinucleotide; NAD+) to mediate a five-step reaction mechanism. Most coenzymes are classified as
vitamins and nutritional deficiencies in some of these important biomolecules can lead to human
disease such as beriberi due to thiamin deficiency, and pellagra, a disease caused by niacin
deficiency which leads to decreased levels of NAD+/NADH.
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