BIOCHEMICAL ENGINEERING

PTT 203

SEMESTER 1
(2012/2013)

PN. NURUL AIN HARMIZA ABDULLAH

 Chapter 5 :
MAJOR METABOLIC
PATHWAYS

PAGE 133. SHULER, M. L. AND KARGI. (2002).

BIOPROCESS ENGINEERING: BASIC CONCEPT.
2 ND E D . U P P E R S A D D L E R I V E R , N J : P R E N T I C E
HALL PTR

COURSE OUTCOME 2:
Ability to categorize the metabolic pathways in microorganisms and analyze the
growth kinetics in both batch and continuous reactors
 CONTENT

5.1 Introduction
5.2 Bioenergetics
5.3 Glucose Metabolism
5.4 Respiration
5.5 Control Sites in Aerobic Glucose Metabolism
5.6 Metabolism of Nitrogenous Compounds
5.7 Nitrogen Fixation
5.8 Metabolism of Hydrocarbons
5.9 Overview of Biosynthesis
5.10 Overview of Anaerobic Metabolism
5.11 Overview of Autotrophic Metabolism
5.12 Summary
 5.1 INTRODUCTION

 Metabolism is the collection amylase

of enzyme catalyzed reactions Starch  smaller glucose
that convert substrates that are external internal
external to the cell into various
internal products.

 Metabolic pathways are

series of chemical reactions
(metabolism) occurring within
a cell.

• Genetic Engineering allows for the alteration of metabolism by insertion or

deletion of selected genes in a predetermined manner (Metabolic
Engineering).
• An understanding of metabolic pathways in the organism of interest is of
primary importance in bioprocess development.

Why we need to learn the metabolic pathways?

 Characteristics of Metabolism

 Varies from organisms to organism.

 Many common characteristics.

 Affected by environmental conditions.

 O2 availability: Saccharomyces cerevisiae
 Aerobic growth on glucose → more cells
 Anaerobic growth on glucose → ethanol

Is S.cerevisiae a
obligate aerobes or
facultative anaerobes?
 Types of Metabolism
Which one is EXERGONIC and
 Catabolism which one is ENDERGONIC ?
 Metabolic reactions in the cell that degrade a

substrate into smaller / simpler products.

 Glucose → CO2 + H2O
 Produces energy.

 Anabolism
 Metabolic reactions that result in the
synthesis of larger / more complex
molecules.
 Glucose → glycogen
 Requires energy.
 5.2 BIOENERGETICS

 It is the quantitative study of the energy relationships and energy

conversions in biological systems.
 It concerned with the energy involved in making and breaking of chemical
bonds in the molecules found in biological organisms.
 All organisms need free energy to keep themselves alive and functioning.
 The Sun is the ultimate energy source for the life processes on earth.
 The source of energy is just one; solar energy. Only plants use that energy
directly. What the organisms use is the chemical energy in the form of foods.
The very first conversion of solar energy into a chemical energy is the
sugar molecule.
 On one side the conversion of solar energy into chemical energy with the help
of photosynthesis happens, and on the other hand this photosynthesis makes it
possible with the passage of time on earth to accumulate free oxygen in the
earth's atmosphere making possible the evolution of respiration. Respiration is
important for bioenergetics as it stores the energy to form a molecule ATP;
adenosine triphosphate. This molecule is a link between catabolism and
anabolisms. The process of photosynthesis is helpful in understanding the
principles of energy conversion i.e. bioenergetics.

What is bioenergetics ?
Plants make their
own food by
photosynthesis.
Carbon dioxide and
water react together
in the presence of
light and
chlorophyll to make
glucose and oxygen.
The glucose is
converted into
starch, fats and oils
for storage. It is
used to make
cellulose for cell
walls, and proteins
for growth and
repair. It is also
used by the plant to
release energy by
respiration.

Respiration and photosynthesis are the main processes dealing with bioenergetics
Please read this…
 Metabolic Reactions

 Can be classified into 3 major categories (refer to figure 5.1 pg 135):

1. Degradatation of nutrients
2. Biosynthesis of small molecules (amino acid, nucleotides)
3. Biosynthesis of large molecules
This reaction takes place in the cell simultaneously.
Figure 5.1: Classes of Reactions (Pg. 135)
Which Class is CATABOLISM and which is ANABOLISM?

Energetics of bacterial growth: balance of anabolic and catabolic reactions.

Figure 5.1: Classes of Reactions (Pg. 135)

Energetics of bacterial growth: balance of anabolic and catabolic reactions.

 ATP : Metabolic Energy

 Adenosine triphosphate (ATP) stored and

transports ENERGY in cells.
 It is the energy currency of life.
 It is used by the cell as „money‟.
 Some activities such as breaking down
glucose produce ATP (money) others such
as making DNA consume ATP ATP
 It contains high-energy phosphate
bonds.
 The energy in ATP is obtained from the
breakdown of foods.
 ATP : Metabolic Energy

 ATP: Adenosine triphosphate and ADP: Adenosine diphosphate.

 So when a phosphate is lost, energy is released.

 Technically speaking, the whole enchilada from a biological

perspective is thus:
 ADP is the end-product that results when ATP loses one of its
phosphate groups located at the end of the molecule. The conversion
of these two molecules plays a critical role in supplying energy for
many processes of life.
 The deletion of one of ATP’s phosphorus bonds generates
approximately 7.3 kilocalories per Mole of ATP.
 ADP can be converted, or powered back to ATP through the process
of releasing the chemical energy available in food; in humans this is
constantly performed via aerobic respiration in the mitochondria.”
ATP : Metabolic Energy
 ATP : Metabolic Energy

 Analog compounds of ATP (GTP, UTP and CTP) also store and transfer
high-energy phosphate bonds but not to the extent of ATP.
 High-energy phosphate compounds (phosphoenol pyruvate and 1,3-
diphosphoglycerate) produced during metabolism, transfer their ~P
group into ATP.
 Energy stored in ATP is later transferred to lower-energy phosphate
compounds (glucose -6-phosphate and glycerol-3-phosphate) – refer
Figure 5.2 pg 135.
5.3 GLUCOSE METABOLISM
Consists of 3 phases:

1. EMP or Glycolysis pathway

• Fermentation of glucose to
pyruvate.

2. Krebs (TCA) or Citric acid cycle

• Conversion of pyruvate to CO2
and NADH.

3. Respiratory or Electron transport

• Formation of ATP by
transferring e- from NADH to
an electron acceptor.

Glycolysis is the breakdown (catabolism)

of glucose to pyruvate under aerobic
conditions
 THE REACTIONS OF GLYCOLYSIS

There are 10 reactions catalyzed by 10 different enzymes

1. Hexokinase: First ATP Utilization

2. Phosphoglucose Isomerase
3. Phosphofructokinase: Second ATP Utilization
4. Aldolase
5. Triose Phosphate Isomerase
6. Glyceraldehyde-3-Phosphate Dehydrogenase: First “High Energy”
Intermediate Formation
7. Phosphoglycerate Kinase: First ATP Generation
8. Phosphoglycerate Mutase
9. Enolase: Second “High energy” Intermediate Formation
10. Pyruvate Kinase: Second ATP Generation
 Where is this
GLYCOLYSIS
took place in a
cell?

Glycolysis
 Summary on Glycolysis:

• The overall reaction of glycolysis is:

Glucose + 2NAD+ + 2ADP + 2Pi 2NADH + 2Pyruvate + 2ATP + 2H2O + 4H+

• The reaction occurs in 10 enzymatically catalysed reactions.

• 3 of the 10 reactions are non-equilibrium, which ensure the pathway go

forward:
 Reaction 1: Glucose to G6P by HK
 Reaction 3: F6P to FBP by PFK
 Reaction 10: PEP to pyruvate by PK
 The Three Products of Glycolysis
1. ATP
• 2 ATP per molecule of glucose were invested and subsequently 4ATP were generated by
substrate-level phosphorylation, giving a net yield of 2ATP per glucose
• ATP produced satisfies most of the cell‟s energy needs.

2. NADH
What is NAD ?
• 2 NAD+ are reduced to 2 NADH
• Reduced NADH represent a source of free energy that can be recovered by subsequent
oxidation
• Under aerobic condition, electron pass from reduced coenzymes thru‟ a series of electron
carriers to the final oxidizing agent, O2, in a process known as electron transport
• The free energy of electron transport drives the synthesis of ATP from ADP
• In aerobic organism, the sequence of events also serves to regenerate oxidized NAD+
• Under anaerobic conditions, NADH must be reoxidized by other means in order to keep the
glycolytic pathway supplied with NAD+

3. PYRUVATE
• 2 pyruvate molecules are produced
• Under aerobic condition, complete oxidation of pyruvate to CO2 and H2O via citric acid cycle
and oxidative phosphorylation, where ATP is generated
• In anaerobic metabolism, pyruvate is metabolized to a lesser extent to regenerate NAD+, via a
process known as fermentation
 Pyruvate Oxidation. In this reaction, the 3 carbonpyruvate is
converted to a 2-carbon acetyl group attached to a Coenzyme A
configuration. A molecule ofNADH + H+ is generated during this
reaction.
 Pyruvate Dehydrogenase

 catalyzes oxidative decarboxylation of pyruvate, to

form acetyl-CoA. The overall reaction is shown
below.

Pyruvic acid + NAD+ Coenzyme A ----------> Acetyl-CoA + CO2 + NADH

 TCA Cycle

• CAC is a common mode of oxidative degradation in eukaryotes

and prokaryotes.
• CAC is also called tricarboxylic acid (TCA) cycle or Krebs cycle.

• CAC accounts for the major portion of carbohydrates, fatty acids

and amino acids oxidation and generates numerous biosynthetic
precursors, therefore amphibolic,that is, it operates both
catabolically as well as anabolically.
• CAC‟s starting compound is acetyl-CoA, the common
intermediate formed by the breakdown of most metabolic fuels.
What is the
different between
prokaryotes and
eukaryotes?
 Where is this CAC
took place in a
cell?

Citric acid cycle

 Reactions of the citric acid cycle:

1. Citrate synthase
2. Aconitase
3. Isocitrate dehydrogenase
4. α-ketoglutarate dehydrogenase
5. Succinyl-CoA synthetase
6. Succinate dehydrogenase
7. Fumarase
8. Malate dehydrogenase

Overall reaction:
3NAD++FAD+GDP+Pi+Acetyl-CoA+H2O 3NADH+3H++FADH2+GTP+CoA+2CO2
 The energy generating capacity of CAC

 The oxidation of one acetyl group to two molecules of CO2 is a four

electron pair process
 For every acetyl-CoA that enters the CAC, 3 molecules of NAD+ are
reduced to NADH, which accounts for 3 electron pairs and one molecule
of FAD is reduced to FADH2 which accounts for the fourth electron pair
 In addition one GTP is produced
 The electrons carried by NADH and FADH2 are funneled into the
electron-transport chain, which culminates with the reduction of O2 to
H2O.
 The energy of electron transport is conserved in the synthesis of ATP by
oxidative phosphorylation
 For every NADH that passes its electron on, approx 3 ATP are produced
from ADP + Pi
 For every FADH2, approx 2 ATP are produced
 Thus one turn of CAC ultimately generates approx 12 ATP
•When glucose is converted to 2
molecules of pyruvate by glycolysis, 2
molecules of ATP are generated and 2
molecules of NAD+ are reduced
In eucaryotic cells, acetyl CoA is produced in the mitochondria from
molecules derived from sugars and fats. Most of the cell's oxidation reactions
occur in these organelles, and most of its ATP is made here.
The three stages of cellular metabolism lead from food to waste
products in animal cells. This series of reactions produces ATP, which is then
used to drive biosynthetic reactions and other energy-requiring processes in the
cell. Stage 1 mostly occurs outside cells––although special organelles called
lysosomes can digest large molecules in the cell interior. Stage 2 occurs mainly in
the cytosol, except for the final step of conversion of pyruvate to acetyl groups on
acetyl CoA, which occurs in mitochondria. Stage 3 occurs in mitochondria.
5.4 RESPIRATION
Electron transport an oxidative phosphorylation

 The ETC gets its name from the fact electrons are
transported to meet up with oxygen from respiration at the
end of the chain.

 The process of forming ATP from ETC is known as

oxidative phosphorylation.
 The Electron Transport Chain

The electron transport chain consists of 3 complexes of

integral membrane proteins:
 the NADH dehydrogenase complex (I)

 the cytochrome c reductase complex (III)

 the cytochrome c oxidase complex (IV)

ATP synthesis is not an energetically favorable reaction: energy is needed in order for it to occur. This energy is derived from the
oxidation of NADH and FADH2 by the four protein complexes of the electron transport chain (ETC). The ten NADH that enter the
electron transport originate from each of the earlier processes of respiration: two from glycolysis, two from the transformation of
pyruvate into acetyl-CoA, and six from the citric acid cycle. The two FADH2 originate in the citric acid cycle.
The events of the electron transport chain involve NADH and FADH, which act as electron transporters as they flow through the
inner membrane space. In complex I, electrons are passed from NADH to the electron transport chain, where they flow through
the remaining complexes. NADH is oxidized to NAD in this process. Complex II oxidizes FADH, garnering still more electrons for
the chain. At complex III, no additional electrons enter the chain, but electrons from complexes I and II flow through it. When
electrons arrive at complex IV, they are transferred to a molecule of oxygen. Since the oxygengains electrons, it is reduced to water.
While these oxidation and reduction reactions take place, another, connected event occurs in the electron transport chain. The
movement of electrons through complexes I-IV causes protons (hydrogen atoms) to be pumped out of the intermembrane space
into the cell cytosol. As a result, a net negative charge (from the electrons) builds up in the matrix space while a net positive charge
(from the proton pumping) builds up in the intermembrane space. This differential electrical charge establishes an electrochemical
gradient. As we will see in the next section, it is this gradient that drives ATP synthesis in oxidative phosphorylation.
 Oxidative phosphorylation

 We now move our discussion past

complexes I-IV on to complex V, called
oxidative phosphorylation, in which ATP
is synthesized from ADP and phosphate
in the matrix of the mitochondria. The
enzyme that catalyzes this reaction is
called proton translocating ATP synthase
the protein component of complex V.

As we introduced in the last section, as a result of the electron transport chain, an electrochemical gradient is
formed on either side of the inner mitochondrial membrane. The outside of the membrane is positive while the
inside is negative. The positive hydrogen ions are allowed to flow back across the membrane through specialized
channels manned by proton translocating ATP synthase, which uses the energy created by the energetically
favorable transport to synthesize ADP and phosphate into ATP.
The transport of just two electrons through the electron transport chain generates enough free energy in the
form of electrochemical gradient to drive the synthesis of one molecule of ATP. The synthesis of ATP necessitates
the dissolution of the electrochemical gradient, however, since the whole process is driven by positive hydrogen
ions (protons) flowing back into the matrix space from the intermembrane space. The ETC maintains the
electrochemical gradient by continuing to generate hydrogen ions.
In total, the process started through the glycolysis of one glucose molecule yields about 32 ATP in oxidative
phosphorylation. In total, oxidative phosphorylation accounts for around 90 percent of the body's total ATP.
 Conclusion on Glucose Metabolism

 We have now
concluded our study of
cell respiration,
following the entire
process of a glucose
molecule from the
cytosol
and glycolysis into the
mitochondria and
through the electron
transport chain and
oxidative
phosphorylation. With
our new knowledge,
we can now produce
an updated version of
our overall map of cell
metabolism
•When glucose is converted to 2
molecules of pyruvate by glycolysis, 2
molecules of ATP are generated and 2
molecules of NAD+ are reduced
•These NADH molecules yield approx 6
ATP on passing their electrons to the
electron transport chain
•When 2 pyruvate molecules are
converted to 2 acetyl-CoA by PDC, the
two molecules of NADH produced in
that process also eventually give rise to 6
ATP
•Two turns of CAC (one for each acetyl
group) generate 24 ATP
•Thus one molecule of glucose can
potentially yield 38 ATP under aerobic
conditions
•In contrast 2 ATP are produced per
glucose under anaerobic conditions
 CONTENT

5.1 Introduction
5.2 Bioenergetics
5.3 Glucose Metabolism
5.4 Respiration
5.5 Control Sites in Aerobic Glucose Metabolism
5.6 Metabolism of Nitrogenous Compounds
5.7 Nitrogen Fixation
5.8 Metabolism of Hydrocarbons
5.9 Overview of Biosynthesis
5.10 Overview of Anaerobic Metabolism
5.11 Overview of Autotrophic Metabolism
5.12 Summary
5.5 CONTROL SITES IN AEROBIC
GLUCOSE METABOLISM
 Control in Glycolysis:
Feedback Inhibition

 The major control site in glycolysis is the phosphorylation of

fructose-6-phsophate by phosphofructokinase:
fructose-6-phosphate + ATP  fructose-1,6-diphosphate + ADP

 Phosphofructokinase (PFK) catalyzes the rate-limiting step in glycolysis

and is the most important control point.

 The enzyme phosphofructokinase is an allosteric enzyme activated

by ADP and Pi but inactivated by ATP. Explain HOW is the mechanism?
ATP
phosphofructokinase (active) phosphofructokinase (inactive)
ADP

When ATP levels are high in the cell, the cell no longer needs metabolic energy production to occur. In this case, PFK's activity is inhibited by allosteric regulation by ATP itself, closing the valve
on the flow of carbohydrates through glycolysis. Recall that allosteric regulators bind to a different site on the enzyme than the active (catalytic) site. Thus ATP binds in two places on PFK: in the
active site as a substrate and in the regulatory site as a negative modulator. ATP bound in the regulatory site acts as a modulator by lowering the affinity of PFK for its other substrate, fructose-6-
phosphate.
Rate-limiting enzyme
for glycolysis is
phosphofructokinase

The phosphorylation of
fructose 6-phosphate is
highly exergonic and
irreversible, and
phosphofructokinase,
the enzyme that catalyzes it,
is the key enzyme in
glycolysis.

Glycolysis
 Control in Glycolysis:
Pasteur Effect

 The Pasteur effect is an inhibiting effect of oxygen on

the fermentation process (Louis Pasteur, 1857).
 The rate of glycolysis under anaerobic conditions is higher than
that under aerobic conditions.
 In the presence of O2, ATP yield is high since the TCA cycle and ETC are
operating.
 So, ADP and Pi become limiting and phosphofructokinase becomes
inhibited.
 A high NADH/NAD+ ratio also reduces the glycolysis rate.
 Control in TCA Cycle:
Feedback Inhibition

 Certain enzymes of the Krebs cycle are also regulated by feedback

inhibition.
 Pyruvate dehydrogenase is inhibited by ATP, NADH, and Acetyl
CoA and activated by ADP, AMP, and NAD+.
 Citrate synthase is inhibited by NAD+.
 In general, high ATP/ADP and NADH/NAD+ ratios reduce the
processing rate of the TCA cycle. Explain HOW?
Citric acid cycle
 5.6 METABOLISM OF NITROGENOUS
COMPOUNDS

 Most organic nitrogen compounds have an oxidation level between

carbohydrates and lipids.
 Nitrogeneous compounds can be used as nitrogen, carbon, and energy
source.
 Proteins are hydrolyzed to peptides and further to amino acids by
proteases.
 Amino acids are first converted to organic acids by deamination
(removal of amino group).
 Deamination Reaction

 Deamination reaction may be oxidative, reductive, or dehydrative,

depend on the enzyme system involved.
 A typical oxidative deamination reaction is as in Eq. 5.10 (pg 143).
 Ammonia released from deamination is utilized in protein and nucleic
acid synthesis as a nitrogen source.
 And organic acids can be further oxidized for energy production (ATP).
 Transamination Reaction

 Is another mechanism for conversion of amino acids

to organic acids and other amino acids.
 The amino group is exchanged for the keto group of
α-keto acid.
 A typical transamination reaction is in Eq.5.11
(pg.143).
 Nitrogen Fixation

 Certain microorganisms fix atmospheric nitrogen to

form ammonia under reductive or microaerophilic
conditions.
 Eg.: Azotobacter, Azotomonas, Azotococcus.
 Nitrogen fixation is catalyzed by the enzyme
“nitrogenase”.
 Metabolism of Hydrocarbons

 This metabolism require oxygen but only few

organisms can metabolize hydrocarbons.
 The first step in metabolism of hydrocarbons is
oxygenation by oxygenases.
 Hydrocarbon molecules alcohol  aldehyde 
organic acid  acetyl-CoA  TCA cycle.
 Overview of Biosynthesis

 Pentose-phosphate pathway:
 Produce significant reducing power  energy supply to cell
 Important for biosynthesis process and this energy is used for
the anabolism processes.
 The main component of biosynthesis is the
production of amino acids.
 The cell must be able to synthesize lipids and
polysaccharides (fatty acids). The key precursor is
acetyl-CoA.
 If the carbon source energy source < 6 carbons, EMP
pathway needs to be operated in reverse reactions.
Gluconeogenesis pathway
with key molecules and
enzymes. Many steps are the
opposite of those found in
theglycolysis.
 Overview of Anaerobic Metabolism

 The production of energy in the absence of oxygen

can be accomplished by anaerobic respiration.
 Use the same pathways as in aerobic metabolism but
differ in the use of an alternative electron acceptor.
 Eg.: Nitrate, NO -3

 Many organism grow without using the ETC. the energy

generated without ETC is called FERMENTATION.
 Since no electron transport is used, the organic substrate
must undergo a balanced series of oxidative and reductive
reactions.
 Overview of Autorophic Metabolism

 Autotrophs obtain their carbon from CO2.

 Calvin cycle provides the building block for
autotrophs growth.
 Photosysthesis.