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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 ofenergy (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. Thispathway yields only two more molecules of ATP per molecule of glucose over the two resulting from glycolysis and ofcourse is the major pathway in wine-making.
b) Glyceropyruvic fermentationDuring 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 oxidativephosphorylation. 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.
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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 (3rd Edition) page 995.
Glycolysis: Glucose to Fructose-1,6-bisphosphate
O
OH
H
HO
OH
HO
O
OH
H
HO
OH
HO
HOH2C
ATP ADP
hexokinase
phosphoglucoisomerase
CH2OH
OH
OH
OHO
Glucose
Glucose-6-phosphate
Fructose-6-phosphateFructose-1,6-bisphosphate
OP
O
O
O
OP
O
O
O
OH
OH
OHO
OP
O
O
O
O P
O
O
O
ATPADP
phosphofructokinase-1
Figure 2
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:
CH2OH
O
HO H
OHH
OHH
CH2OH
HO H
OHH
OHH
CH2OH
OH
OHH
HO H
OHH
OHH
CH2OH
OHH
H O
keto-enol
tautomerism
keto-enol
tautomerism
D-fructoseD-glucose
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Glycolysis: Fructose-1,6-bisphosphate to Pyruvate
Fructose-1,6-bisphosphate
OH
OH
OHO
OP
O
O
O
O P
O
O
O
HO OPO3
O H OPO3
O
OH
Glyceraldehyde-3-phosphateDihydroxyacetone phosphate
aldolase
triose-phosphate isomerase
O3PO OPO3
O
OH
1,3-bisphosphoglycerate
NAD
NADH
glyceraldehyde-3-phosphate
dehydrogenase
O OPO3
O
OH
O OH
O
OPO3
3-phosphoglycerate
phosphoglycerate kinase
2-phosphoglyceratephosphoenolpyruvate
enolase
phosphoglycerate mutase
O
O
OPO3
ATP ADP
H2O
O
O
O
pyruvate
pyruvate kinase
ATP
ADP
2
2
222
2 2
Figure 3
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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 (3rd Edition) page 984.
Remembering that an aldol is the reaction of an enolate (anion of an aldehyde or ketone) with an aldehyde or ketone:
HO OPO3
O H OPO3
O
OH
CH2OPO3
O
HO H
OHH
OHH
CH2OPO3
Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate Fructose-1,6-bisphosphate
2
2
2
2
The enzymic retroaldol activates the carbonyl of fructose by forming its imine outlined below:
H OH
CH2OPO3
H OH
HHO
O
CH2OPO3
H OH
CH2OPO3
H O
HHO
HN
CH2OPO3
H2N enzyme
S
imine formation
with -amino groupof lysine of
triose phosphate isomerase
enzyme
SH
H OPO3
O
OH
Glyceraldehyde-3-phosphate
HHO
HN
CH2OPO3
enzyme
S
H OH
CH2OPO3
H O
HHO
N
CH2OPO3
enzyme
SHHO
O
CH2OPO3
HH
H
H
Dihydroxyacetone phosphate
HO OPO3
O
2
2 2 2
2
2
2
22
2
enamineimine
As with the glucose-fructose conversion, dihydroxyacetone phosphate is readily converted via enol formation toglyceraldehyde 3-phosphate:
HO OPO3
O
H OPO3
O
OH
HO OPO3
OH
Dihydroxyacetone phosphateGlyceraldehyde-3-phosphate
2 2
2
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).
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O OPO3
O
OH
1,3-bisphosphoglycerate
H OPO3
O
OH
Glyceraldehyde-3-phosphate
NAD NADH
glyceraldehyde-3-phosphate
dehydrogenase
PO
O
OO OPO3
O
OH
HPO42
22
Nicotinamide adenine dinucleotide (NAD+) is the oxidizing agent. NAD is covered in Organic Chemistry, Bruice (3rd
Edition) page 996.
O
OH
O
N
NN
N
NH2
OH
PO
O
O
OH
O
OH
PO
ON
NH2
OO
N
NH2
O
R
N
NH2
O
R
H HH
H
NAD + H + 2e NADH
nicotinamide adenine dinucleotide, NAD
Next, this energy is given up to an ATP by transfer of the phosphoryl group of the acyl phosphate.
O3
PO OPO3
O
OH
1,3-bisphosphoglycerate
O OPO3
O
OH
3-phosphoglycerate
phosphoglycerate kinase
ATPADP
22 2
The last phase of glycolysis transforms 3-phosphoglycerate into pyruvate. The remaining phosphate group is transferred
from carbon-3 to carbon-2.
O OPO3
O
OH
O OH
O
OPO3
3-phosphoglycerate 2-phosphoglycerate
phosphoglycerate mutase2 2
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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 OH
O
OPO3
2-phosphoglycerate phosphoenolpyruvate
enolase
O
O
OPO3
H2O
O
O
Opyruvate kinase
ATPADP
2 2
O
O
OH
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.
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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
lactate dehydrogenase
NADH NAD
O
O
OH
lactate
In alcoholic fermentation, pyruvate is first decarboxylated to acetaldehyde (ethanal) using pyruvate decarboxylase. Themechanism for this reaction is covered in Organic Chemistry, Bruice (3rd Edition) page 1005.
O
O
OO
H
pyruvateethanal
This enzyme requires Mg2+ and the cofactor, thiamine pyrophosphate, TPP. Thiamine or vitamin B1 has the structure and
TPP is obvious:
N
N
N
S
NH2
OH
N
N
N
S
NH2
O P
O
O
O P
O
O
O
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?
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The Alcoholic Fermentation Pathway
Fructose-1,6-bisphosphate
OH
OH
OHO
OP
O
O
O
O P
O
O
O
HO OPO3
OH OPO3
O
OH
Glyceraldehyde-3-phosphateDihydroxyacetone phosphate
aldolase
triose-phosphate isomerase
O3PO OPO3
O
OH
1,3-bisphosphogrycerate
NAD
NADH
glyceraldehyde-3-phosphate
dehydrogenase
O OPO3
O
OH
O OH
O
OPO3
3-phosphoglycerate
phosphoglycerate kinase
2-phosphoglycerate
phosphoenolpyruvate
enolase
phosphoglycerate mutase
O
O
OPO3
ATP
ADP
H2O
O
O
O
pyruvate
pyruvate kinase
ATP ADP
OH
O
ethanol
ethanal or acetaldehyde
alcohol dehydrogenase
pyruvate decarboxylase
CO2
H
FIGURE 4
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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 giveethanal and thiamine pyrophosphate.
The mechanism for the decarboxylation follows:
O
O
O
S
N
R1
R2
S
N
R1
R2
HO
O
O
H
S
N
R1
R2
CO2
H
S
N
R1
R2
O
H
H
S
N
R1
R2 B
O
H
HO
pyruvate thiamine pyrophosphateactive acetaldehyde, TPP-C2
thiamine pyrophosphateethanal hydroxyethyl thiamine pyrophosphate
The second step reduces ethanal into ethanol by NADH.
O
H
ethanal
HO
ethanol
alcohol
dehydrogenase
From an energy viewpoint, glycolysis followed by alcoholic fermentation supplies the yeast with two molecules of ATP per
molecule of glucose.
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b)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-3-
phosphate, 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-1,6-
bisphosphate, 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 moleculesundergo 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 theglyceropyruvic 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 strictlyrespiratory 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.
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The Glyceropyruvic Fermentation Pathway
Fructose-1,6-bisphosphate
OH
OH
OHO
OP
O
O
O
O P
O
O
O
HO OPO3
OH OPO3
O
OH
Glyceraldehyde-3-phosphateDihydroxyacetone phosphate
aldolase
O3PO OPO3
O
OH
1,3-bisphosphoglycerate
NAD
NADH
glyceraldehyde-3-phosphate
dehydrogenase
O OPO3
O
OH
O OH
O
OPO3
3-phosphoglycerate
phosphoglycerate kinase
2-phosphoglycerate
phosphoenolpyruvate
enolase
phosphoglycerate mutase
O
O
OPO3
ATP
ADP
H2O
O
O
O
pyruvate
pyruvate kinase
ATP ADP
HO OPO3
OH
Secondary products
(-ketoglutaric acid, succinic acid,
butanediol, diacetyl, acetoin, etc)
HO OH
OH
Glycerol-3-phosphate
Glycerol
FIGURE 5
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Pathways for the Formation of Acetic Acid in Yeasts
Pyruvate
Ethanal
Ethanol
Acetate
Acetyl CoA
NADPHNADP
NADHNAD
NAD
NADH
CO2
CO2HSCoA
H2O
HSCoA
HSCoA
Lipid synthesis
FIGURE 6
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 yeastproduces during fermentation. Sweet wines (including ice wine) made from musts with high sugar concentrations haveelevated 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 2,3-
butanediol (Figure 7).
Acetoin, Diacetyl and 2,3-Butanediol Formation by Yeasts in Anaerobiosis
Pyruvate TPP-C2 -Acetolactate
PyruvateTPP
CO2
O
O
OH
O
OH
OH
Diacetyl Acetoin 2,3-Butanediol
NAD
NADH
CO2CO2
NADNADH
NADHNAD
FIGURE 7
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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 -acetolactate. See
the following mechanism.
O
O
O
S
N
R1
R2S
N
R1
R2
HO
O
O
H
S
N
R1
R2
CO2 HO
S
N
R1
R2
HOO
O
O
active acetaldehyde
or TPP-C2pyruvate thiamine pyrophosphate
TPP
pyruvate TPP-C2
HS
N
R1
R2
O
OH
O
OH
B
S
N
R1
R2
O
O
OH
O
-acetolactate TPP
-Acetolactate can either undergo oxidative decarboxylation to form diacetyl or a nonoxidative decarboxylation to formacetoin. 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 oxidativedecarboxylation 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.
HOOCCOOH
OH
COOH
O
H
O
OH
Malate Pyruvate Ethanal Ethanol
Malic enzyme Pyruvate decarboxylase Alchol dehydrogenase
NAD NADH
CO2
CO2NADNADH
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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
S
N
R1
R2
S
N
R1
R2
HO
O
O
H
S
N
R1
R2
CO2 HO
pyruvate thiamine pyrophosphate active acetaldehyde, TPP-C2
S
N
R1
R2
HO
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.
active acetaldehyde, TPP-C2
SS
NH(CH2)4E2
O
lipoate
S NR1
R2
HO
S NR1
R2
S
NH(CH2)4E2
O
O
HH B
B
S
NH(CH2)4E2
O
S NR1
R2
O
NH(CH2)4E2
O
CoASH
SHSH
HS
HS
SCoA
O
SS
NH(CH2)4E2
O
FAD E3
FADH2 E3 FAD E3
NAD NADH
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 Krebs Cycle. This cycle is the final common pathway for fuel molecules aminoacids, 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 (3rd Edition) page 994.
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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.
O2C
O
CO2
Oxaloacetate
H2O
O2C
CO2
OH
SCoA
OH
enol form of
acetyl-SCoA
SCoA
O
CO2
OH
CO2
CO2
Citrate
O2C
CO2
OH
O
O
Citryl-SoA
citrate
synthase
citrate
synthase
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).
CO2
OH
CO2
CO2
Citrate
CO2
CO2
CO2
CO2
CO2
CO2
HO
H2O
cis-Aconitate
H2O
Isocitrate
aconitase aconitase
Step 3 consists of the oxidative decarboxylation of isocitrate to yield -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 synthesizeapproximately 2.5 ATP.
CO2
CO2
CO2
HO
Isocitrate
CO2
CO2
O
Oxalosuccinate
CO2
CO2
O
-ketoglutarate
O
OHB
isocitrate
dehydrogenase
isocitrate
dehydrogenase
NADHCO2
NAD
Step 4 is another reversible oxidative decarboxylation (G' = 34 kJ/mol) reaction. NAD+ and CoASH react with -
ketoglutarate to yield succinyl-SCoA, CO2 and NADH. This step is catalyzed by the -ketoglutarate dehydrogenasecomplex, which is very similar to the pyruvate dehydrogenase complex and requires TPP, FAD, lipoic acid and Mg2+.
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CO2
CO2
O
-ketoglutarate
SCoA
CO2
O
succinyl-SCoA
CoASHketoglutarate
dehydrogenase
complex
NADHNAD
TPP, FAD, lipoic acid
CO2
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).
SCoA
CO2
O
succinyl-SCoA
GDP GTP
succinyl-SCoA
synthetase O2C
CO2
succinate
Step 6 is a reversible, stereospecific oxidation reaction (G' = 0 kJ/mol) catalyzed by the succinate dehydrogenase
complex.
O2C
CO2
succinate
O2C
CO2
fumarate
FAD FADH2
succinate
dehydrogenase
complex
Step 7 is the reversible, stereospecific hydration of fumerate to give L-malate, catalyzed by fumerase (G' = 4 kJ/mol).
O2C
CO2
fumarate
O2C
CO2
L-malate
OHfumerase
H2O
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).
O2C
CO2
L-malate
OH
NADHNAD
malate
dehydrogenaseO2C
CO2
oxaloacetate
O
The 4-carbon oxaloacetate condenses with the 2-carbon acetyl CoA to form 6-carbon citrate. An isomer of citrate is thenoxidatively decarboxylated. The resulting 5-carbon -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 arelost 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.
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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 levelphosphorylation during the formation of succinate.
The respiration of the same amount of sugar produces 18 to 19 times more biologically usable energy (ATP) available toyeasts than fermentation.
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The Citric Acid Cycle
O2C
O
CO2
OH
CO2
CO2
OH
CO2
CO2
O2C
CO2
CO2
CO2
CO2
CO2
CO2
HO
CO2
CO2
CO2
O
CO2
CO2
O
SCoA
CO2
O
O2C
CO2
CO2
O2C
CO2
O
SCoA
O
oxidative decarboxylation
NAD NADH
HSCoA CO2pyruvate acetyl CoA
H2O
HSCoA
Oxaloacetate
Citrate
H2O
cis-Aconitate
H2O
Isocitrate
Oxalosuccinate
NADNADH
-ketoglutarate
CO2
NAD
NADH
Succinyl CoA HSCoA
CO2
Succinate
GDP
GTP
HSCoA
Fumerate
FAD
FADH2
Malate
H2O
NADH
NAD
Respiratory chain
and
ATP production
pyruvate dehydrogenase
citratesynthase
aconitase
isocitr
ate
dehy
drog
enase
a-ketoglutaratedehydrogenasecomplex
succinyl-CoA
synthetase
succinatedehydrogenase
fumerase
malate
dehydrogenase
ADP
ATP
FIGURE 8