Bichemistry of Yeast Fermentation - Sugars (1)

7/29/2019 Bichemistry of Yeast Fermentation - Sugars (1)

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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


H OPO3

O

OH

Glyceraldehyde-3-phosphate

NAD NADH


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


O OPO3

O

OH

3-phosphoglycerate


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


OH

OH

OHO

OP

O

O

O

O P

O

O

O

HO OPO3

OH OPO3

O

OH


aldolase

triose-phosphate isomerase

O3PO OPO3

O

OH

1,3-bisphosphogrycerate

NAD

NADH


dehydrogenase

O OPO3

O

OH

O OH

O

OPO3

3-phosphoglycerate


2-phosphoglycerate

phosphoenolpyruvate

enolase


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


OH

OH

OHO

OP

O

O

O

O P

O

O

O

HO OPO3

OH OPO3

O

OH


aldolase

O3PO OPO3

O

OH


NAD

NADH


dehydrogenase

O OPO3

O

OH

O OH

O

OPO3

3-phosphoglycerate


2-phosphoglycerate

phosphoenolpyruvate

enolase


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+.


16/18

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.


17/18

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.


18/18

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

Documents

Bichemistry of Yeast Fermentation - Sugars (1)