Metabolic Stoichimetry and

Energetics
 Introduction
• A living cell is a complex chemical reactor in which more than 1000
independent enzyme catalyzed reactions occur
• The total of all chemical reaction activities which occur in the cell is
called metabolism.
• Metabolic reactions tend to be orgainized into sequence called
metabolic pathways.
• A cell produces order ( itself and its offspring) from its disorderly
surrounding .
• Energy from the environment is used to drive the metabolic process
 Introduction
Three types of metabolism
- Aerobic: Use free oxygen
-Anaerobic: Do not use free oxygen
-facultative anaerobes : A third class of cells can grow in either environment
and known as facultative anaerobes. Yeast is a familiar example of this metabolic
variety
Two different kinds of energy are tapped by inhabitants of microbial world
-Light : Organism which relay on light are called phototrophs

-Chemical : While chemotrophs extract energy by breaking down certain

nutrients.
 Introduction
• Further chemotrophs is subdivided as follows
• Lithotrophs: Oxidize inorganic materials
• Organotrophs: Employ organic nutrients for energy production
• The energy obtained from the environment is typically stored and shuttled in
convenient high-energy intermediates such as ATP. The cell uses this energy to
perform three types of work:
• chemical synthesis of large or complex molecules (growth)
• transport of ionic and neutral substances into or out of the cell or its
internal organelles
• mechanical work required for cell division and motion.
• All these processes are (by themselves) nonspontaneous and result in an
increase of free energy of the cell. They occur when simultaneously couple to
another process which has a negative free-energy change of greater magnitude.
Schematic overview of important KEGG metabolic pathways in bacteroids
• In order to grow and reproduce, cells must ingest the raw materials
necessary to manufacture membrane, protein, walls, chromosomes and
other components
• Four major requirements are evident: carbon, nitrogen, sulfur and
phosphorus
• Reactions within the cell have been subdivided into three classes:
• degradation of nutrients
• biosynthesis of small molecules
• biosynthesis of large macromolecules
• Each reactions are catalyzed by an enzyme. The enzyme serve the essential
function of determining which reaction occur and their relative rates
 Thermodynamics Principles
• To get an idea of whether a certain reaction in the cell will run forward or
backword, we will use a number of approximation since full analysis of
metabolic network is not practical. First we consider the free-energy
change of a chemical reaction

αA  βB  γC  δD
This equation can be written in the form

c d  γ δ
ΔG  ΔG  RTln  α β 
0'

a b 
Where, ∆G0' is the standard free energy change
Contd…. Thermodynamics Principles
• In a closed system, the reaction will proceed left to right if and only if ∆G' is
negative. Accordingly, ∆G' is zero at equilibrium give the following
relationship:
G   RT ln Keq
0'

• Where 
ceq deq
K eq   
aeq beq
• If water or H+ are involve in the reaction, their concentrations do not enter
into the calculation of the right hand side in equation. The value already
includes the water and H+ concentration (for pH 7)
Contd….
Thermodynamics Principles

• Consider the reaction between two isomers in the Embden-Mayerhof

pathway for glucose breakdown

CHO
CH2 OH
CHOH
C O
∆G0'= -1830 cal/mol
CH2O P
CH2 O P
Glyceraldehyde
3-phosphate Dihydroxyacetone-P

• Where P denotes phosphate. Because of the negative free energy

change, equilibrium favors the dihydroxyacetone by a 22:1 ratio.
Contd….
Thermodynamics Principles
• Many biological reaction and energy conversion process involve oxidation-
reduction reaction such as

Aox  Bred  A red  Box

• This type of reaction is described using the standard potential change
0 0 0
ΔE  E Aox Ared  E BBox Bred 
Where E0A ox Ared  is the standard half-cell potential for the half reaction


Aox  2e  Ared
Contd….
Thermodynamics Principles

• As a reference point for half-cell potential value, the hydrogen half-cell (at
pH=0) is assigned a value of zero:

2H  2e  H 2 E 0  0.000V (pH  0)

• The free energy change and corresponding potential changes are related by

G  nFE
• Where n is the number of electrons transferred and F is equal to 23.062
kcal/V mol
 Anabolic and Catabolic Reactions

• Anabolic reactions use ATP to combine smaller molecues such as glucose

, into larger compounds such as glycogen and release small amounts of
heat

• Catabolic reactions break down larger molecules into smaller compounds

and release energy in the form of ATP and heat
 Metabolic Reaction Coupling: ATP and NAD
• The enzymatic hydrolysis of ATP to yield ADP and inorganic phosphate has
a large negative free-energy change

ATP + H2O  ADP + Pi G0’ = -7.3 kcal/mol

Where Pi indicate inorganic phosphate

• A substantial amount of free-energy may be released by the hydrolysis

• By reversing the reaction and adding the phosphate to ADP, free energy can
be stored for late use
-Another example from Embden-Meyerhof-Parnas pathway serves to illustrate the concept of a
common chemical intermediate. Conversion of an aldehyde in an aqueous medium to a
carboxylic acid results in a free energy decrease of about 7000 cal/mol.
-In biochemical glucose oxidation, when 3-phosphoglyceraldehyde is converted into a
carboxylic acid (3-phosphoglycerate), ATP is simultaneously regenerated from ADP.
-The free energy decrease resulting from aldehyde oxidation is coupled by the cell enzymes to
the simultaneous regeneration of ATP.
- The elementary reaction sequence by which the conversion actually occur is shown in
reaction below.
1. Oxidation of aldehyde to carboxylic acid
RC H O  H 2 O  2 H  RC O O  H  
 G 10   7 kcal/mol
2. Same reactions, coupled to ATP generation (glucose oxidation)
3  4 0
RC H O  H P O 4  ADP  2 H  RC O O  A TP  G   0 kcal/mol
2-
2
3. Evidently, 2-1 yields,
3  4 0
ADP  HPO 4  H  ATP  H 2O  G   7 kcal/mol
2-
3
The central important features of the last two reaction shown in previous slides are
1. The appearance of a common intermediate; the same compounds is a product of
the first reaction and a reactant in the second and
2. The phosphorylated intermediate formed and consumed has a larger free energy
of hydrolysis (with phosphate removal) than ATP.

Example : The elementary reaction occurs in 2 ( previous slides) are:

O- H H O O- H H O O-
-
-
O P O C C C H + HPO 4
=
2H + H 2O + O P O C C C O P O-
O H OH O H OH O

O- H H O O-
-
O P O C C C O P O- + A D P 3- R COO- + A T P 4-
O H OH O
• Thus glucose metabolism is the process at which cell generates the ATP needed for
endergonic process
• This generation is accomplished by the conversion of a partially metabolized nutrient into a
high-energy phosphorylated intermediate, which then donates a phosphate to ADP via an
enzyme-catalyzed reaction
• It provides a useful means of storing considerable fractions of free energy of fuel oxidation.
Free energies of hydrolysis of several called phosphate donors are greater than ∆G0’ for ATP
hydrolysis.
Example, phosphoenolpyruvate ∆G0’ = -14.8 kcal mol-1.
1,3-diphosphoglycerate ∆G0’ = -11.8 kcal mol-1

Hydrolysis of this compounds can be used to drive ADP phosphorylation

• Similarly, ATP hydrolysis serves to phosphorylate “low energy” phosphate compounds.

Example, glucose-6-phosphate ∆G0’ = -3.3 kcal mol-1
glycerol-1-phosphate ∆G0’ = -2.2 kcal mol-1
 Oxidation and Reduction: Coupling via NAD
• Oxidation-reduction reactions are conducted biologically and the connection
between these mechanisms and ATP metabolism.

• Oxidation of a compound means that it loses electron and that addition of

electron is reduction of a compound.

• When an organic compound is oxidized biologically, it usually loses electrons in

the form of hydrogen atoms: consequently, oxidation is synonymous with
dehydrogenation.

• Similarly, hydrogenation is the usual way of adding electron or reducing a

compounds, e.g. the reduction of pyruvic acid and oxidation of lactic acid
CH3 + 2H (reduction CH3
of pyruvic acid) HC OH
C O
COOH + 2H (oxidation COOH
of lactic acid)
Pyruvic Lactic
acid acid
 NH2
N N

N N
O O
H H
H H
OH H
HO P O
O Nicotinamide adenine dinucleotide (NAD)
OH P O
CONH2

N
O O
H H
H H
OH H
• Pairs of hydrogen atoms freed during oxidation or required in reductions are carried by
nucleotide derivatives, especially nicotinamide adenine dinucleotide (NAD) and its
phosphorylated form of NADP. These compounds were classified as coenzymes since they
usually must be present when an oxidation or reduction is conducted.
• When hydrogen atoms are needed, for example, the nicotinamide group of reduced NAD can
contribute them by undergoing the oxidation.
• This oxidation is readily reversible, so that NAD can also accept electron ( H atom) when they
are made available by oxidation of other compounds.
• Oxidized and reduced forms of NAD in denoted by NAD+ and NADH.
NADH NAD+
H H H
O O
C - 2H (oxidation) C
HC C NH2 HC C NH2
HC CH + 2H (reduction) HC CH
N N

R R
Reduction form Oxidation form
- When a metabolite is oxidized, NAD+ accepts two electrons plus a hydrogen
ion (H+) and NADH results.

NADH then carries energy to cell for other uses

• NAD and related pyridine nucleotide compounds carrying hydrogen also
participate in ATP formation in aerobic metabolism. The hydrogen atoms in
NADH are combined with oxygen in a cascade of reactions known as the
respiratory chain. The energy released in this oxidation is sufficient to form
three molecule of ATP from ADP.
• All the biological systems, e.g., anaerobic, aerobic, or photosynthetic
metabolism, utilize ATP as central means of accumulating oxidative or radiant
energy for driving the endergonic processes of the cell.
 Carbon metabolism

• Breakdown of nutrients to obtain energy is called catabolism.

• Carbohydrates are by far the most important class of carbonaceous nutrients
for fermentation, although some microbial species can also utilize amino acids,
hydrocarbons and other compounds.
• There are at least seven glucose fermentation pathways and the particular one
used and the end products produced depend on the microorganism involved
 Embden-Meyerhof-Parnas Pathway

• Embden-Meyerhof-Parnas Pathway involved in ten enzyme catalyzed steps

which start with glucose and end with pyruvate.

• The EMP steps involve isomerization, ring splitting, or transfer of a small

group such as hydrogen or phosphate.

• Two moles of pyruvate are produced per mole of glucose passing through the
pathway.
Embden-Meyerhof-Parnas Pathway contd…….

ATP hydrolysis coupled with two reactions and each reaction involve sufficiently negative free
negative energies to drive ADP phosphorylation.
CH2OH CH2OPO32-
CH2OPO32-
O H H O H
H O CH2OH
ATP ADP H
H H
OH H OH H OH
Phosphohexoisomerase
OH Hexokinase OH OH OH
OH
H
H OH H OH OH H
Glucose Glucose 6-phosphate Fructose 6-phosphate
ATP
Phospho-
f ructokinase

2-
ADP
CH 2OPO32- CH2OPO3
HC O
Triose isomerase O CH2OPO32-
HCOH C O Aldolase
H OH
CH 2OPO3 2- CH 2OH
Glyceraldehyde Dihydroxyacetone OH
H
3-phosphate phosphate
OH H
Fructose 1,6-diphosphate
Embden-Meyerhof-Parnas Pathway contd…….

CH 2OPO 32-
HC O ADP ATP CH 2OPO32-
NAD+ NADH HCOH
HCOH HCOH
2-
C OPO3 3-phosphoglycerate
CH 2OPO32- Glyceraldehyde 3-phosphate kinase COO-
dihydrogenase O
Glyceraldehyde 1,3-Diphosphoglycerate 3-Phosphoglycerate
6-phosphate

Phosphoglyceramutase

H 2O
ATP CH 2OH
CH3 ADP CH2
HCOPO 32-
C O C O PO 32-
Pyruvate kinase
-
E n o l a se COO-
COO COO-
Pyruvate Phosphoenolpyruvate 2-Phosphoglycerate
The overall stoichiometry of the EMP pathway is
C6H12O6 + 2 Pi + 2 ADP + 2 NAD+ 2 C3H4O3 + 2 ATP + 2 (NADH + H+)
- Stored chemical energy and reducing power result from overall pathway. Energy storage
accomplished by this or other substrate rearrangement pathways is called substrate-level
phosphorylation
- In muscle cell and lactic acid bacteria, the reactions of the EMP are followed by single step
C3H4O3 + NADH + H+ C3H6O3 + NAD+
- This overall reaction sequence from glucose to lactic acid is called glycolysis. It is interesting
to compare the free energy change for glycolysis

Glucose + 2 Pi + 2 ADP 2 lactose + 2 ATP + 2 H2O G0’ = -32,400 cal/mol

-With corresponding quantity for the glucose breakdown alone

Glucose 2 lactose G0’ = -47,000 cal/mol

-A total free-energy of 14.6 kcal or 7.3 kcal for each mole of ATP generated has been conserved
by the pathway as high energy phosphate compounds
 Other carbohydrate catabolic pathway:
Pentose phosphate pathway
• The pathway begins with the glycolytic intermediate glucose 6-P.
• It reconnects with glycolysis because two of the end products of the pentose
pathway are glyceraldehyde 3-P and fructose 6-P; two intermediates further
down in the glycolytic pathway.
• It is for this reason that the pentose pathway is often referred to as a shunt.
Also known as:
• Pentose shunt
• Hexose monophosphate shunt
• Phosphogluconate pathway
 Pentose phosphate pathway

• The pathway yields reducing potential in the form of NADPH to be used in

anabolic reactions requiring electrons.
• The pathway yields ribose 5-phosphate.
• Nucleotide biosynthesis leading to:
• DNA
• RNA
• Various cofactors (CoA, FAD, SAM, NAD+/NADP+).
 Other carbohydrate catabolic pathway:
Pentose phosphate pathway

• Pentose phosphate cycle or pathway ( also called the hexose monophosphate or

shunt) begins by oxidizing glucose phosphate

Glucose 6- phosphate + NADP+ 6-phosphogluconate + NADPH +H+

• The major function of the pentose phosphate pathways is supplying the cell
with NADPH which in turn carries electrons to biosynthesis reaction
Fig. general scheme of the pentose
phosphate pathway

Ref: principle of biochemistry

NADPH + H+ is formed
from two separate
reactions.

The glucose 6-phosphate

DH (G6PD) reaction is
the rate limiting step and
is essentially irreversible.

Cells have a greater need

for NADPH than ribose 5-
phosphate.
 Pentose phosphate pathway
• The total reaction scheme is quite complicated, but its overall result is indicated
by the following summary of pentose phosphate pathway stoichiometry:

Glucose + 12 NADP+ +7H2O +ATP 6 CO2 + Pi + 12(NADPH +H+) +ADP

• The net effect is complete oxidation of one mole of glucose 6-phosphate

liberating CO2 and transferring all of the electrons (H) to NADP.
• This overall reaction consumes ATP. In order to provide ATP to the cell, an
incomplete pentose phosphate cycle may occur with overall stoichiometry

2 glucose + 6NADP+ + ATP 2fructose 6-phosphate + glyceraldehyde 3-

phosphate +3CO2 + 6(NADPH+H+) +ADP + Pi
 Contd…..

• Pentose phosphate pathway is advantageous in the sense of including ribose 5-

phosphate and erythrose 4-phosphate, important precursors for purine and
pyrimidine biosynthesis, among its intermediates. These components are absent
from the EMP pathways
• As a consequence, microorganisms such as lactobacilli which posses only EMP
pathway require a complex medium including pentose, purines and pyrimidines
when grown in anerobic environment.
• E. coli, on the other hand is able to grow anaerobically in simpler media using
the EMP and pentose phosphate pathways simultaneously ( for example, in the
ratio of 75% EMP: 25% pentose phosphate pathways under certain culture
conditions)
• This illustrate the important general concept that cells may possess multiple
catabolic pathways for a given nutrient and may employ more than one pathway
simultaneously, presumably to optimize growth considering both energy and
precursors requirements.
 Entner-Doudoroff (ED) pathway
• The ED pathway is an unique glycolytic pathway in prokaryotes.
• The ED pathway was first identified in Pseudomonas saccharophila by Entner
and Doudoroff.
• The ED pathway turned out to be the main glycolytic pathway in prokaryotes
that do not possess enzymes of the EMP pathway.
• The ED pathway functions as the main glycolytic pathway in other G (-)
bacteria such as Zymomonas and Azotobacter species.
• Gluconate is metabolized through the ED pathway in some other G(-) bacteria
including Escherichia coli and in some coryneform bacteria.
 Entner-Doudoroff (ED) pathway

• Alternative catabolic pathway from glucose-6-phosphate to smaller

molecules
• Many prokaryotes have another pathway for the degradation of carbohydrates
called the Entner-Doudoroff or ED pathway.
• Found in some bacteria as alternative to normal glycolytic pathway
• Other bacteria that do have glycolytic pathway possess these enzymes as a
side-path
 Entner-Doudoroff (ED) pathway
• The overall stoichiometry of this reaction sequence is

Glucose + ATP + NADP+ glyceraldehyde 3-phosphate + pyruvic acid +

ADP + NADPH + H+

• Two moles of ADP can be phosphorylated by reacting one mole of

glyceraldehyde 3- phosphate to pyruvate through the same reactions as used
for conversion in the EMP pathway, the energy yield of the ED pathways is
relatively poor: one mole ATP per mole of glucose processed.
 Key enzymes of the ED pathway

1 = glucose 6-phosphate dehydrogenase

2 = 6-phosphogluconase dehydratase
3 = 2-keto-3-doexy-6-phsphogluconate
aldolase
4 - as in the EMP pathway

-NAD(P)+-dependent glucose-6-phosphate dehydrogenase converts glucose-6-phosphate to 6-

phosphogluconolactone that is converted to 6-phosphogluconate.
- 6-Phosphogluconate dehydratase removes water molecule from 6-phosphogluconate and produces 2-keto-3-deoxy-
phosphogluconate (KDPG).
- KDPG aldolases splits KDPG into pyruvate and glyceraldehyde-3-phosphate.
- The key enzymes in the ED pathway are 6-phosphogluconate dehydrogenase and KDPG aldolase.
 Respiration
• If an energy-producing process in which organic or reduced inorganic are
oxidized by inorganic compounds.
• When, an oxidant other than oxygen is involved, the process is called
anaerobic respiration, the term anaerobic respiration is being reserved for the
situation typical of eucaryotes and many bacteria where O2 is the oxidant.
• In most common forms of respiration, an organic compound is oxidized using
oxygen.
 TCA cycle
• Tricarboxylic acid (TCA) cycle, also called Krebs cycle or citric acid cycle is
the central metabolic hub of the cell
• It accounts for the majority of carbohydrate, fatty acid and amino acid
oxidation. It also accounts for a majority of the generation of these
compounds and others as well
• Involved 8 reactions catalyzed by following enzymes
• Citrate synthase
• Aconitase
• Iso‐citrate dehydrogenase
• ketoglutarate dehydrogenase
• Succinyl‐Coenzyme A synthetase
• Succinate dehydrogenase
• Fumerase
• Malate dehydrogenase
 Reactions of Citric Acid Cycle
• 1. Citrate synthase: Formation of Citroyl CoA intermediate.
• 2. Binding of Oxaloacetate to the enzyme results in conformational change
which facilitates the binding of the next substrate, the acetyl Coenzyme A.
There is a further conformational change which leads to formation of products.
This mechanism of reaction is referred as induced fit model.
• 2. Aconitase: This enzyme catalyses the isomerization reaction by removing
and then adding back the water (H and OH) to cis-aconitat in at different
positions. In this way produced Isocitrate consume reapidly in next step.
• 3. Isocitrate dehydrogenase: There are two isoforms of this enzymes, one
used NAD+ and other uses NADP+ as electon acceptor. The enzyme
isocitrate dehydrogenase convert isocitrate to alpha-ketoglutarate

6 carbons 5 carbons
4. alpha-ketoglutarate dehydrogenase: This is a complex of different
enzymatic activities similar to the pyruvate dehydrogenase complex.
The alph-ketoglutarate is converted to Succinyl-CoA by enzyme called
alpha-ketoglutarate dehydrogenase

5 carbons 4 carbons
5. Succinyl CoA synthatase: Succinyl CoA , likely Acetyl CoA has a thioester
bond with very negative free energy of hydrolysis .In this reaction, the
hydrolysis of the thioester bond leads to the formation of phosphoester bond with
inorganic phosphate. This phosphate is transferred to Histidine residue of the
enzyme and this high energy, unstable phosphate is finally transferred to GDP
resulting in the generation of GTP
6. Succinate Dehydrogenase: Oxidation of succinate to fumarate.
This is the only citric acid cycle enzyme that is tightly bound to the inner
mitrochondrial membrane. It is an FAD dependent enzymes
7. Fumarase: Hydration of fumarate to malate: It is a highly sterospecific
enzyme.
8. L-malate dehydrogenase: oxidation of malate to oxaloacetate: It is an
NAD+ dependent enzyme. Reaction is pulled in forward direction by the next
reaction (citrate synthase reaction) as the oxaloacetate is depleted at a very
fast rate
Over all reaction cycle