BIOCHEMISTRY OF YEAST FERMENTATION Sugar ... - People

BIOCHEMISTRY OF YEAST FERMENTATION
The synthesis of living material is endergonic, requiring the consumption of energy. Most animals, bacteria, fungi including
yeast are chemoorganotrophs, they draw their energy from the oxidation of organic nutrients. Chlorophyllous plants
(phototrophs) collect solar energy and store this energy in the form of reduced organic compounds.
In a growing organism, energy produced by degradation reactions (catabolism) is transferred to a chain of synthesis
reactions (anabolism).
The first law of thermodynamics (∆E = q + w) tells us that only part of this energy is converted to useful work (the rest is
dissipated as heat). The free energy that is converted to work can be used for transport, movement or synthesis. In most
cases the free energy transporter is adenosine triphosphate (ATP). Hydrolysis of ATP to ADP releases 7.3 kcal.mol -1 of
energy (using the biochemical standard state with a pH of 7 instead of 1.0).
Yeasts obtain their ATP from the oxidation of sugars.
Sugar Degradation pathways
There are three pathways yeast (usually Saccharomyces cerevisiae) can obtain energy through the oxidation of glucose and
Figure 1 outlines these pathways:
a) Alcoholic fermentation under anaerobic conditions (no oxygen).
The pyruvate resulting from glycolysis is decarboxylated to acetaldehyde (ethanal) which is reduced to ethanol. This
pathway yields only two more molecules of ATP per molecule of glucose over the two resulting from glycolysis and of
course is the major pathway in wine-making.
b) Glyceropyruvic fermentation
During wine-making 8% of glucose follows this pathway and it is important at the beginning of the alcoholic fermentation
of grape must when the concentration of alcohol dehydrogenase (required to convert ethanal to ethanol) is low.
c) Respiration under aerobic conditions (presence of oxygen). Glycolysis of glucose yields pyruvate and two molecules of
ATP per molecule of glucose. Pyruvate is then oxidized to carbon dioxide and water via the citric acid cycle and oxidative
phosphorylation. This pathway yields a further 36-38 molecules of ATP per molecule of glucose and obviously the yeast
would prefer this route. However the amount of oxygen is carefully controlled during the wine-making process and this
pathway is forbidden!
All three pathways start with the initial stage of glycolysis, the conversion of glucose into fructose-1,6-bisphosphate.

Glycolysis
This series of reactions transforms glucose into pyruvate with the formation of 2 molecules of ATP.
Hexose (glucose) is transported across the plasmic membrane into the cytosol of the cell moving with the concentration
gradient (concentrated outer medium to dilute inner medium).
The first stage of glycolysis converts glucose into fructose-1,6-bisphosphate and requires two molecules of ATP, see Figure
2. Glycolysis is covered in Organic Chemistry, Bruice (3 rd Edition) page 995.
O
O
P
O
HO
O
HOH 2 C
HO
Glycolysis: Glucose to Fructose-1,6-bisphosphate
OH
Glucose
O
O
OH
OH
H
Fructose-1,6-bisphosphate
OH
OH
O
O P
O
O
ATP ADP
ADP
hexokinase
ATP
phosphofructokinase-1
Figure 2
O
O
O
P
O
HO
O
P
O
O
HO
OH
O
OH
H
Glucose-6-phosphate
O
OH
phosphoglucoisomerase
O
OH
Fructose-6-phosphate
The first and third steps involve adding phosphates to carbons 1 & 6 which are endogonic and require energy (ATP). The
second step is the isomerization of glucose into fructose which proceeds by enol formation:
H O
H OH
HO H
H OH
H OH
CH2OH D-glucose
keto-enol
tautomerism
H
OH
OH
HO H
H OH
H OH
CH2OH keto-enol
tautomerism
CH2OH O
HO H
H OH
H OH
CH2OH D-fructose
OH
CH 2 OH

Glycolysis: Fructose-1,6-bisphosphate to Pyruvate
O
O
P
O
O
OH
O
OH
OH
Fructose-1,6-bisphosphate
O
O P
HO
2
OPO3 O
O triose-phosphate isomerase H
2
OPO3 Dihydroxyacetone phosphate
O
2
O OPO3 OH
3-phosphoglycerate
O
aldolase
phosphoglycerate kinase
O
O
OH
glyceraldehyde-3-phosphate
dehydrogenase
Glyceraldehyde-3-phosphate
O
NAD
NADH
OH
1,3-bisphosphoglycerate
O OH
O
enolase
2 2
OPO3 OPO3 2-phosphoglycerate
phosphoglycerate mutase
ATP ADP
H 2O
Figure 3
2
2
O3PO OPO3 O
phosphoenolpyruvate
ADP
ATP
O
O
O
pyruvate
pyruvate kinase

The second stage of glycolysis forms pyruvate, see Figure 3.
First fructose-1,6-bisphosphate is cleaved to glyceraldehyde 3-phosphate. This is a retroaldol condensation (or a reverse
aldol condensation) and consequently the enzyme is called aldolase. The mechanism for this reaction is covered in Organic
Chemistry, Bruice (3 rd Edition) page 984.
Remembering that an aldol is the reaction of an enolate (anion of an aldehyde or ketone) with an aldehyde or ketone:
2
CH2OPO3 HO
O
2
OPO3 H
O
OH
2
OPO3 HO
H
H
O
H
OH
OH
2
CH2OPO3 Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate Fructose-1,6-bisphosphate
The enzymic retroaldol activates the carbonyl of fructose by forming its imine outlined below:
2
CH2OPO3 O
HO H
H OH
H OH
2
CH2OPO3 H 2N enzyme
S
imine formation
with ε-amino group
of lysine of
triose phosphate isomerase
2
CH2OPO3 H
N enzyme
HO H
H O H S
H OH
2
CH2OPO3 2
CH2OPO3 2
CH2OPO3 2
CH2OPO3 O
H
N enzyme
H
N enzyme
HO H
HO H S
HO H
H
H
H S
Dihydroxyacetone phosphate
imine
enamine
2
HO OPO3 O
H O
H OH
2
CH2OPO3 Glyceraldehyde-3-phosphate
O
2
H OPO3 As with the glucose-fructose conversion, dihydroxyacetone phosphate is readily converted via enol formation to
glyceraldehyde 3-phosphate:
2 2
HO OPO3 HO OPO3 O
Dihydroxyacetone phosphate
OH
O
OH
2
H OPO3 OH
Glyceraldehyde-3-phosphate
The enzyme is triose phosphate isomerase (a triose is a 3 carbon suger). The equilibrium is driven to the RHS as
glyceraldehyde-3-phosphate is rapidly removed by subsequent reaction. In other words a molecule of glucose yields two
molecules of glyceraldehyde-3-phosphate.
The third phase of glycolysis comprises two steps which recover part of the energy from glyceraldehyde-3-phosphate.
Initially the aldehyde is oxidized to a carboxylic acid (∆G°′=-43 kJ.mol -1 ) and this energy is trapped in a phosphate bond of
the mixed anhydride of the carboxylic acid and phosphoric acid (∆G°′= +49 kJ.mol -1 ).

O
2
H OPO3 OH
Glyceraldehyde-3-phosphate
glyceraldehyde-3-phosphate
dehydrogenase
NAD NADH
O
2
O OPO3 OH
2
HPO4 O
O
P
O
O
O
OPO3 OH
1,3-bisphosphoglycerate
Nicotinamide adenine dinucleotide (NAD + ) is the oxidizing agent. NAD is covered in Organic Chemistry, Bruice (3 rd
Edition) page 996.
O
O
O
P
O
P
O
O
O
OH
OH
O
O
OH
OH
N
N
N
NH 2
N
O
N
NH 2
nicotinamide adenine dinucleotide, NAD
H
N
R
O
NH 2
H
H H
NAD + H + 2e NADH
Next, this energy is given up to an ATP by transfer of the phosphoryl group of the acyl phosphate.
O
ADP ATP
O
2
O3PO OH
OPO
2
3
phosphoglycerate kinase
O
OH
OPO
2
3
1,3-bisphosphoglycerate
3-phosphoglycerate
The last phase of glycolysis transforms 3-phosphoglycerate into pyruvate. The remaining phosphate group is transferred
from carbon-3 to carbon-2.
O
O
phosphoglycerate mutase
O OPO
2
3
O 2
OH
OH
OPO 3
3-phosphoglycerate 2-phosphoglycerate
N
R
O
NH 2

The 2-phosphoglycerate loses a molecule of water, yielding the enol, phosphoenolpyruvate. Phosphoenolpyruvate then
transfers its phosphate group to ADP, producing a second ATP and after a keto-enol isomerism, pyruvate.
O
H 2O
O OH
O
2
OPO3 enolase
2
OPO3 2-phosphoglycerate phosphoenolpyruvate
O
ADP
ATP
pyruvate kinase
O
O
OH
O
O
O
pyruvate
Glycolysis produces four ATP molecules; cleavage of fructose-1,6-bisphosphate produces two molecules of glyceraldehyde
3-phosphate and oxidation of each glyceraldehyde 3-phosphate to pyruvate produces two molecules of ATP.
Two molecules of ATP are immediately used to activate a new molecule of glucose and the net gain of glycolysis is
therefore two ATP molecules per molecule of glucose metabolized.

a) Alcoholic Fermentation
Oxidation is the loss of electrons and these electrons must be passed on to an electron acceptor or oxidizing agent. This
oxidizing agent in fermentation is nicotinamide adenine dinucleotide NAD + and at some stage in the process the reduced
oxidizing agent, NADH, must pass the electrons on and be reoxidized.
The terminal electron acceptor is acetaldehyde which is reduced to ethanol while the NADH is oxidized back to NAD + and
able to continue the glycolysis cycle by oxidizing another glyceraldehyde-3-phosphate. See figure 4.
In humans the terminal electron acceptor is pyruvate which is reduced to lactate in muscles (stiffness).
O
O
O
pyruvate
NADH NAD
lactate dehydrogenase
In alcoholic fermentation, pyruvate is first decarboxylated to acetaldehyde (ethanal) using pyruvate decarboxylase. The
mechanism for this reaction is covered in Organic Chemistry, Bruice (3 rd Edition) page 1005.
O
O
O
pyruvate
ethanal
This enzyme requires Mg 2+ and the cofactor, thiamine pyrophosphate, TPP. Thiamine or vitamin B1 has the structure and
TPP is obvious:
N
NH 2
N
N
S
OH
N
NH 2
N
O
N
O
H
O
OH
lactate
thiamine thiamine pyrophosphate
The aromatic benzene ring with two nitrogens is called a pyrimidine and the five membered ring containing a sulfur and a
nitrogen is called a thiazole, is this ring aromatic? The hydrogen on the carbon between the nitrogen and sulfur is acidic,
why?
S
O
O P
O
O
O P
O
O

O
ethanol
H
O
OH
O
pyruvate
alcohol dehydrogenase
O
ethanal or acetaldehyde
CO 2
pyruvate decarboxylase
The Alcoholic Fermentation Pathway
O
O
P
O
HO OPO 3
O
O
Dihydroxyacetone phosphate
ATP ADP
pyruvate kinase
NAD
NADH
O
O
OH
O
OH
Fructose-1,6-bisphosphate
aldolase
OH
triose-phosphate isomerase
OPO 3
phosphoenolpyruvate
FIGURE 4
O
O P
O
O
O
phosphoglycerate kinase
enolase
H OPO 3
OH
Glyceraldehyde-3-phosphate
glyceraldehyde-3-phosphate
dehydrogenase
O
O 3 PO OPO 3
OH
1,3-bisphosphogrycerate
O
phosphoglycerate mutase
H 2 O
O OPO 3
O
OH
ADP
ATP
3-phosphoglycerate
O OH
OPO 3
2-phosphoglycerate

First pyruvate condenses with thiamine pyrophosphate to form an addition compound, which readily decarboxylates to form
“active acetaldehyde” or TPP-C2. Protonation then gives hydroxyethyl thiamine pyrophosphate which breaks down to give
ethanal and thiamine pyrophosphate.
The mechanism for the decarboxylation follows:
O
O
O
H
S
N
R 1
R 2
pyruvate thiamine pyrophosphate
The second step reduces ethanal into ethanol by NADH.
O
H
O
S
O
HO
N
R 1
R 2
S
N
R 1
R 2
CO 2
B
H
HO
H
H
O
S
S
N
R 1
R 2
active acetaldehyde, TPP-C 2
ethanal thiamine pyrophosphate
hydroxyethyl thiamine pyrophosphate
O
H
ethanal
alcohol
dehydrogenase
From an energy viewpoint, glycolysis followed by alcoholic fermentation supplies the yeast with two molecules of ATP per
molecule of glucose.
HO
ethanol
N
R 1
R 2

) Glyceropyruvic Fermentation
At the beginning of alcoholic fermentation of grape must, the pyruvate decarboxylase and alcohol dehydrogenase are
weakly expressed. The concentration of acetaldehyde is low and NADH looks for another terminal acceptor so that it can be
reoxidized to react with another molecule of glyceraldehyde-3-phosphate.
In glyceropyruvic fermentation, dihydroxyacetone phosphate picks up the electrons and gets reduced to glycerol-3phosphate,
which is dephosphorylated into glycerol. See Figure 5.
In this fermentation only two molecules of ATP are produced for every molecule of glucose oxidized, as only one molecule
of glyceraldehyde-3-phosphate forms for each molecule of glucose.
Since two molecules of ATP are required to activate the glucose in the first steps of glycolysis in yielding fructose-1,6bisphosphate,
the net gain in ATP in glyceropyruvic fermentation is zero and there is no biologically assimilable energy for
yeasts.
Wines contain about 8g glycerol per 100g ethanol. During grape must fermentation, about 8% of the sugar molecules
undergo glyceropyruvic fermentation and 92% undergo alcoholic fermentation. The fermentation of the first 100g of
glucose forms the majority of the glycerol, after which glycerol production slows, but never stops. Glyceropyruvic
fermentation is therefore more than an inductive fermentation which generates NAD + when ethanal is not yet present.
Alcoholic and glyceropyruvic fermentations overlap slightly throughout fermentation.
Glycerol has a sugary flavor similar to glucose; however in wine the sweetness of glycerol is practically imperceptible. The
secondary products decrease wine quality and consequently the wine-maker would wish to limit the extent of the
glyceropyruvic fermentation.
Pyruvic acid is derived from glycolysis and in glyceropyruvic fermentation it does not form ethanal and ethanol (the NADH
is used to reduce dihydroxyacetone) and thus goes on to form secondary products, such as succinic acid, diacetyl etc.
Secondary Products
Succinic Acid
Aerobic respiration is carried out in the mitochondria and during fermentation (alcoholic and glyceropyruvic) they are not
functional. However the enzymes of the citric acid cycle are present in the cytoplasm. In these anaerobic conditions, the
citric acid cycle cannot be completed since the succinodehydrogenase activity requires the presence of FAD, a strictly
respiratory coenzyme. The chain of reactions is therefore interrupted at succinate, which accumulates (0.5-1.5 g/L). The
NADH generated by this portion of the citric acid cycle (oxaloacetate to succinate) is reoxidized by the formation of
glycerol from dihydroxyacetone.
Acetic Acid
Acetic acid is the principle volatile acid in wine. It is produced during bacterial spoilage but is always formed by yeasts
during fermentation. Beyond a certain limit, which varies depending on the wine, acetic acid has a detrimental organoleptic
effect on wine quality. In healthy grape must with a moderate sugar concentration (less than 220 g/L, Sacch. cerevisiae
produces relatively small quantities (100-300 mg/L).
The biochemical pathway for the formation of acetic acid in wine yeasts has not been clearly identified. The hydrolysis of
acetyl CoA will produce acetic acid as will aldehyde dehydrogenase by the oxidation of ethanal. Figure 6 shows the
pathways used by yeast to form acetic acid.

Secondary products
(α-ketoglutaric acid, succinic acid,
butanediol, diacetyl, acetoin, etc)
O
O
O
pyruvate
The Glyceropyruvic Fermentation Pathway
O
O
P
O
HO OPO 3
O
O
Dihydroxyacetone phosphate
HO OPO 3
pyruvate kinase
OH
ATP ADP
Glycerol-3-phosphate
HO OH
OH
Glycerol
O
O
OH
O
OH
Fructose-1,6-bisphosphate
OPO 3
NAD
NADH
phosphoenolpyruvate
FIGURE 5
aldolase
OH
O
O P
O
O
O
phosphoglycerate kinase
enolase
H OPO 3
OH
Glyceraldehyde-3-phosphate
glyceraldehyde-3-phosphate
dehydrogenase
O
O 3 PO OPO 3
OH
1,3-bisphosphoglycerate
O
phosphoglycerate mutase
H 2O
O OPO 3
O
OH
ADP
ATP
3-phosphoglycerate
O OH
OPO 3
2-phosphoglycerate

CO 2
Pyruvate
Ethanal
Ethanol
NAD
NADH
Pathways for the Formation of Acetic Acid in Yeasts
NAD
HSCoA
NADP
NADH
CO 2
NADPH
HSCoA
FIGURE 6
Acetyl CoA
Acetate
H 2 O
HSCoA
Lipid synthesis
The practical wine-making conditions that lead Sacch. cerevisiae to produce abnormally high quantities of acetic acid are
fairly well known. The higher the sugar concentration in the grape must, the more acetic acid (and glycerol) the yeast
produces during fermentation. Sweet wines (including ice wine) made from musts with high sugar concentrations have
elevated acetic acid levels.
Lactic Acid
Lactic acid is another secondary product of fermentation. It is derived from pyruvic acid, directly reduced by yeast
lacticodehydrogenase. In alcoholic fermentation, the yeast synthesizes predominately D(-) lacticodehydrogenase and form
200-300 mg/L of D(-) lactic acid.
Wines that have undergone malolactic fermentation can contain several grams per litre exclusively of L(+) lactic acid.
Acetoin, Diacetyl and 2,3-Butanediol
Yeasts also make use of pyruvic acid to produce acetoin (2-hydroxybutan-2-one), diacetyl (butan-2,3-dione) and 2,3butanediol
(Figure 7).
Acetoin, Diacetyl and 2,3-Butanediol Formation by Yeasts in Anaerobiosis
TPP
Pyruvate
Pyruvate TPP-C 2 α-Acetolactate
CO 2
CO 2
O
O
NAD
NADH
NADH
NAD
CO 2
OH
O
Diacetyl Acetoin 2,3-Butanediol
FIGURE 7
NAD
NADH
OH
OH

Pyruvate condenses with thiamine to form active acetaldehyde, TPP-C2 (see decarboxylation of pyruvate under alcoholic
fermentation) which condenses with a second molecule of pyruvate, and kicks off the thiamine to form α-acetolactate. See
the following mechanism.
O
O
O
O
pyruvate
O
O
H
H
HO
S
N
S
R 1
R 2
pyruvate TPP-C 2
thiamine pyrophosphate
TPP
N
R 1
R 2
O
O
HO
O
O
S
N
R 1
R 2
OH
O
H
S
N
R 1
R 2
CO 2
B
HO
S
N
R 1
R 2
active acetaldehyde
or TPP-C 2
O
O
O
OH
S
N
R 1
R 2
α-acetolactate TPP
α-Acetolactate can either undergo oxidative decarboxylation to form diacetyl or a nonoxidative decarboxylation to form
acetoin. Acetoin can also form by reduction of diacetyl. The reversible reduction of acetoin forms 2,3-butanediol.
Yeasts produce diacetyl from the start of alcoholic fermentation. Reduction to acetoin and 2,3-butanediol takes place in the
days that follow the end of the fermentation., when wines are conserved on yeast biomass.
Acetoin and particularly diacetyl are strong smelling compounds which evoke a buttery aroma. The concentrations of these
compounds from alcoholic fermentation are a few milligrams per litre, which is below their thresholds.
Degradation of Malic acid
Saccharomyces cerevisiae degrades malic acid to an extent of about 10-15% during alcoholic fermentation. The oxidative
decarboxylation is performed by malic enzyme. The resulting pyruvate is decarboxylated to ethanal which is reduced to
ethanol.
Grape must contains approximately 5 g/L of malic acid.
HOOC
COOH
CO 2
Malic enzyme
COOH Pyruvate decarboxylase H Alchol dehydrogenase
OH NAD NADH O
CO2 O NADH
NAD
Malate Pyruvate Ethanal Ethanol
OH

c) Respiration
When yeast has plenty of oxygen (aerobic conditions) it follows the respiratory pathway. Respiration takes place in the
mitochondria, while alcoholic fermentation takes place in the cytosol of the cell. Pyruvate (originating from glycolysis in
the cytosol) forms acetyl-CoA via an oxidative decarboxylation in the presence of coenzyme A (CoA) and NAD + . Pyruvate
dehydrogenase in the interior of the mitochondria, catalyzes this reaction using the cofactors, thiamine pyrophosphate, TPP,
lipoamide, flavin-adenine dinucleotide, FAD and NAD + .
The pyruvate dehydrogenase system is a group of three enzymes responsible for the conversion of pyruvate to acetyl CoA.
The first enzyme in the system catalyzes the condensation of thiamine pyrophosphate, TPP, with pyruvate to form an
addition compound, which readily decarboxylates to form “active acetaldehyde” or TPP-C2.
O
O
O
H
S
N
R 1
R 2
O
O
HO
S
N
R 1
R 2
pyruvate thiamine pyrophosphate active acetaldehyde, TPP-C 2
The second enzyme of the system (E2) requires lipoate, a coenzyme that becomes attached to its enzyme by forming an
amide with the amino group of lysine. The disulfide bond of lipoate is cleaved when it undergoes nucleophilic attack by
TPP-C2. Then TPP is eliminated from the tetrahedral intermediate.
Coenzyme A reacts with the thioester in a transesterification reaction substituting CoA for dihydrolipoate. At this point
acetyl CoA is formed.
The third enzyme oxidizes dihydrolipoate to lipoate with FAD. NAD + then oxidizes the enzyme bound FADH2 back to
FAD.
HO
S N R 1
R2 active acetaldehyde, TPP-C2 O
SCoA
S
SH
S
S
H B
SH
FAD E 3
S
lipoate
O
NH(CH 2 ) 4 E 2
O
O
NH(CH 2 ) 4 E 2
NH(CH 2 ) 4 E 2
CO 2
B
CoASH
HO
H
O
R 2
S
S
N
R 1
R 2
S N R 1
NAD
O
HS
S
NADH
HS
FADH 2 E 3 FAD E 3
R 2
HO
O
S
N
R 1
R 2
NH(CH 2 ) 4 E 2
O
S N R 1
NH(CH 2 ) 4 E 2
The activated acetyl unit, acetyl CoA is then completely oxidized into carbon dioxide by the Citric Acid Cycle, also known
as the Tricarboxylic Acid Cycle (TCA) or Kreb’s Cycle. This cycle is the final common pathway for fuel molecules – amino
acids, fatty acids and carbohydrates. Most fuel molecules enter the cycle as acetyl CoA. See Figure 8. The citric acid cycle
is covered in Organic Chemistry, Bruice (3 rd Edition) page 994.

Step 1 of the TCA cycle involves two reactions: an aldol condensation between acetyl CoA and oxaloacetate to give
citryl_SCoA, and the hydrolysis of citryl-SCoA to yield citrate. The hydrolysis of citryl-SCoA provides the thermodynamic
driving force and makes this step irreversible from a practical standpoint (∆G°′ = −32 kJ/mol). Since both the aldol
condensation and the hydrolysis are catalyzed by citrate synthase, they are treated as a single step.
O 2C
CO 2
O
Oxaloacetate
OH
SCoA
enol form of
acetyl-SCoA
citrate
synthase
O 2C
CO 2
OH O
Citryl-SoA
SCoA
citrate
synthase
H 2 O
O 2C
CO 2
OH O
O
CO2 CO2 OH
CO2 Citrate
Step 2 is also a two phase process; dehydration followed by rehydration. Isocitrate is the final product and aconitase is the
enzyme for this reversible reaction (∆G°' = +6 kJ/mol).
CO 2
CO 2
CO 2
Citrate
OH
H 2O
aconitase
CO 2
CO 2
CO2 cis-Aconitate
H 2O
aconitase
HO
CO 2
CO 2
Isocitrate
Step 3 consists of the oxidative decarboxylation of isocitrate to yield α-ketoglutarate and CO2. NAD + is the oxidizing agent
and oxalosuccinate is an intermediate in this irreversible reaction (∆G°' = −21 kJ/mol). Two of the six CO2 and two of the
NADH produced by the total oxidation of glucose are generated by this step. Each of the NADHs can be used to synthesize
approximately 2.5 ATP.
HO
CO 2
CO 2
Isocitrate
CO 2
NAD
NADH
isocitrate
dehydrogenase
B
H
O
CO 2
CO 2
O
O
Oxalosuccinate
CO 2
isocitrate
dehydrogenase
O
CO 2
CO 2
CO 2
α-ketoglutarate
Step 4 is another reversible oxidative decarboxylation (∆G°' = −34 kJ/mol) reaction. NAD + and CoASH react with αketoglutarate
to yield succinyl-SCoA, CO2 and NADH. This step is catalyzed by the α-ketoglutarate dehydrogenase
complex, which is very similar to the pyruvate dehydrogenase complex and requires TPP, FAD, lipoic acid and Mg 2+ .

O
CO 2
CO 2
α-ketoglutarate
CoASH
NAD
NADH
α−ketoglutarate
dehydrogenase
complex
TPP, FAD, lipoic acid
CO 2
O
CO 2
SCoA
succinyl-SCoA
In step 5, the cleavage of the thioester link in succinyl-CoA drives the phosphorylation of guanosine diphosphate (GDP), a
reversible process (∆G°' = −3 kJ/mol).
O
CO 2
SCoA
succinyl-SCoA
GDP GTP
succinyl-SCoA
synthetase O 2 C
CO 2
succinate
Step 6 is a reversible, stereospecific oxidation reaction (∆G°' = 0 kJ/mol) catalyzed by the succinate dehydrogenase
complex.
O 2C
CO 2
succinate
FAD FADH 2
succinate
dehydrogenase
complex
O 2 C
CO 2
fumarate
Step 7 is the reversible, stereospecific hydration of fumerate to give L-malate, catalyzed by fumerase (∆G°' = −4 kJ/mol).
O 2C
CO 2
fumarate
H 2 O
fumerase
O 2C
CO 2
L-malate
Step 8 is the irreversible oxidation of L-malate by NAD + to regenerate oxaloacetate so that the cycle can start again. The
reaction is catalysed by malate dehydrogenase (∆G°' = +30 kJ/mol).
O 2C
CO 2
OH
L-malate
NAD
NADH
malate
dehydrogenase
O 2C
CO 2
oxaloacetate
The 4-carbon oxaloacetate condenses with the 2-carbon acetyl CoA to form 6-carbon citrate. An isomer of citrate is then
oxidatively decarboxylated. The resulting 5-carbon α-ketoglutarate is oxidatively decarboxylated to yield 4-carbon
succinate and oxaloacetate is regenerated via fumerate and malate.
Two carbons enter the cycle as an acetyl unit and two carbons leave the cycle as carbon dioxide. The oxidation state of the
two carbons of acetyl-CoA is zero, and that of two molecules of carbon dioxide is +8 (CO2 is +4) and so eight electrons are
lost in these oxidations. These electrons are transferred as pairs to 3 NAD + molecules and one FAD molecule. These
electron carriers yield 11 molecules of ATP when they are oxidized by O2 in the electron transport chain (oxidative
phosphorylation). In addition one high energy phosphate bond is formed in each round of the citric acid cycle.
O
OH

The respiration of a glucose molecule produces 36-38 molecules of ATP. Two originating from glycolysis, 28 from the
oxidative phosphorylation of NADH and FADH2 generated by the Krebs cycle and two from substrate level
phosphorylation during the formation of succinate.
The respiration of the same amount of sugar produces 18 to 19 times more biologically usable energy (ATP) available to
yeasts than fermentation.

FAD
H 2 O
fumerase
O 2 C
O 2 C
CO 2
Fumerate
CO 2
Succinate
HSCoA
O 2 C
CO 2
CO 2
OH
The Citric Acid Cycle
O
O
pyruvate HSCoA CO2 acetyl CoA
FADH 2
succinate
dehydrogenase
GTP
succinyl-CoA
synthetase
Malate
ATP
GDP
O
NAD
ADP
CO 2
SCoA
oxidative decarboxylation
pyruvate dehydrogenase
NAD NADH
malate
dehydrogenase
NADH
Succinyl CoA HSCoA
NADH
a-ketoglutarate
dehydrogenase
complex
NAD
O 2 C
CO 2
O
O
Oxaloacetate
CO 2
H 2 O
Respiratory chain
and
ATP production
CO 2
CO 2
α-ketoglutarate
FIGURE 8
SCoA
citrate synthase
CO 2
HSCoA
CO 2
CO2 Citrate
CO 2
OH
O
aconitase
CO 2
CO 2
CO 2
Oxalosuccinate
H 2 O
NADH
HO
CO 2
CO 2
CO 2
cis-Aconitate
CO 2
CO 2
Isocitrate
CO 2
NAD
isocitrate
dehydrogenase
H 2O