Transcript Matabolic Stoichiometry and Energetics in

Matabolic Stoichiometry and
Energetics in Microorganisms
Dr. A.K.M. Shafiqul Islam
Metabolism
 A living cell is a complex chemical reactor in which
more than 1000 independent enzyme-catalyzed
reaction occurs
– The total of all chemical reaction activities which occur
in the cell is called metabolism.
 The metabolic reaction tend to be organized into
sequences called metabolic pathways which
connect one reaction with another
Important Coenzymes




NAD+
NADP+
FAD
Coenzyme A
 A cell produces order from its disorderly
surrounding things
 Energy from the environment is used to drive the
metabolic process
 In bioprocess engineering, the energy exchanges
helps explain the major distinction between cell
function in the presence and absence of oxygen
Types of Metabolism
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.
 Further subdivision of chemotrophs is
possible
– Lithotrophs
 Oxidize organic materials
– Organotrophs
 Employ organic nutrients for energy production
 The energy obtained from the environment is stored and
shuttled in the high-energy intermediates such as ATP.
Cell use this energy to perform three types of work:
– chemical synthesis of large or complex molecules
– transportation of ionic or neutral substances into or out of the cell
or its internal organcells
– 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.
 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
Thermodynamic 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
A  B  C  D
 We can write
 

c
d
0'
G  G  RT ln   
a b
(1)



(2)
 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 realtionship
(3)
G 0'   RT ln Keq
 where
K eq 

ceq
d eq
 
aeq
beq
(4)
 If water or H+ are involve in the reaction, their
concentrations do not enter into the calculation of the right
hand side (4). The value already includes the water and H+
concentration (for pH 7)
 Consider the reaction between two isomers in the
Embden-Mayerhof pathway for glucose breakdown
CHO
CH2 OH
CHOH
CH2 O
C
P
Glyceraldehyde
3-phosphate
G0’= -1830 cal/mol
O
CH2 O
P
Dihydroxyacetone-P
 Where P denotes phosphate. Because of the negative free
energy change, equilibrium favors the dihydroxyacetone by
a 22:1 ratio.
 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
ΔE  E
0
Aox A red 
E
0
BBox Bred 
where E 0A ox Ared  is the standard half-cell potential for the
half reaction
A ox  2e   A red
 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
Metabolic Reaction Coupling:
ATP and NAD
ATP
 Energy is released as food is oxidized
 Used to form ATP from ADP and Pi
ADP + Pi + Energy
ATP
 In cells, energy is provided by the hydrolysis
of ATP
ATP
ADP + Pi + Energy
 The enzymatic hydrolysis of ATP to yield ADP and
inorganic phosphate has a large negative freeenergy 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

Embden-Meyerhof-Parnas pathway serves to illustrate
the concept of a common chemical intermediate
1.
Oxidation of aldehyde to carboxylic acid
RCHO  H 2O  2H  RCOO  H 
G10  7kcal/mol
2.
Same reactions, coupled to ATP generation (glucose
oxidation)
RCHO  HPO 4  ADP 3  2H  RCOO   ATP 4
G 02  0kcal/mol
2-
3. Reaction 2 and 1 yield
ADP 3  HPO 4  H   ATP 4  H 2O
2-
G 30  7kcal/mol
 Example
OH H O
=
O P O C C C H + HPO4
O
H OH
O- H H O
O2H + H2O + -O P O C C C O P OO
H OH
O
OH H O
OO P O C C C O P OO
H OH
O
+ ADP3-
RCOO- + ATP4-
 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
The phosphorylation of various
compounds serves several functions
 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
 Highly ionized organic substances are virtually unable to
permeate the cell’s plasma membranes. The charged
phosphorylated compounds which serves as metabolic
intermediates may therefore be contained within the cell.
Thus maximum amounts of energy and chemical raw
materials can be extracted from a nutrient.
Oxidation 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
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
similarly, hydrogenation is the usual way of adding electron
CH3
C O
COOH
Pyruvic
acid
+ 2H (reduction
of pyruvic acid)
+ 2H (oxidation
of lactic acid)
CH3
HC OH
COOH
Lactic
acid
NH2
N
N
O
N
N
O
H
H
H
OH
H
H
Nicotinamide adenine
dinucleotide (NAD)
HO P O
O
OH P O
CONH2
N
O
O
H
H
H
OH
H
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.
NADH
NAD+
H
H
HC
HC
H
C
N
O
C
CH
R
Reduction form
- 2H (oxidation)
NH2
+ 2H (reduction)
HC
HC
C
N
O
C
CH
R
Oxidation form
NH2

NAD serves two major functions
1. Analog to one of ATP’s job –
reducing power made available during breakdown of
nutrient is carried to biosynthetic reaction. The
reducing power is used for the construction of cell
components.
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 CATABOLISM
 Breakdown of nutrients to obtain energy is called
catabolism.
 Fermentation of carbohydrates, e.g., glucose, are under
this category.
 The 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 (EMP)
 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.
 ATP hydrolysis coupled with two reactions and each
reaction involve sufficiently negative free negative energies
to drive ADP phosphorylation.
CH2OH
H
ATP
O
H
OH
ADP
H
H
Hexokinase
H
OH
CH2OPO32-
H
H
OH
OH
H
OH
OH
Glucose 6-phosphate
Glucose
CH2OH
O
Phosphohexoisomerase
OH
OH
OH
OH
H
H
CH2OPO32O
H
H
OH
H
Fructose 6-phosphate
ATP
Phosphof ructokinase
ADP
CH2OPO32-
CH 2OPO32-
HC O
Triose isomerase
HCOH
CH 2OPO 32-
Glyceraldehyde
3-phosphate
C O
CH 2OH
Dihydroxyacetone
phosphate
CH2OPO32-
O
H
Aldolase
OH
OH
H
OH
H
Fructose 1,6-diphosphate
CH 2OPO 32HC O
NAD+
HCOH
CH 2OPO 32-
Glyceraldehyde
6-phosphate
C OPO32Glyceraldehyde 3-phosphate
dihydrogenase
ATP
ADP
HCOH
NADH
3-phosphoglycerate
kinase
O
1,3-Diphosphoglycerate
CH 2OPO32HCOH
COO3-Phosphoglycerate
Phosphoglyceramutase
H2 O
CH 3
C O
COOPyruvate
ADP
ATP
Pyruvate kinase
CH2
CH 2OH
C O PO 32-
HCOPO 32-
-
COO
Phosphoenolpyruvate
Enolase
COO2-Phosphoglycerate
C6H12O6 + 2 Pi + 2 ADP + 2 NAD+
2 C3H4O3 + 2 ATP + 2 (NADH + H+)
Stored chemical energy and reducing power result from
overall pathway. This is called substrate-level pathway
In muscle cell and lactic acid bacteria, the reactions of the
EMP are followed by single step
C3H4O3 + NADH + H+
C3H6O3 + NAD+
The overall reaction sequence from glucose to lactic acid is
called glycolysis
 Free-energy change for overall glycolysis reaction
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.
Carbohydrate Catabolism
 The breakdown of carbohydrates to release
energy
– Glycolysis
– Krebs cycle
– Electron transport chain
Other Carbohydrate Catabolic
Pathways
 The pentose phosphate cycle or pathway begins by
oxidizing glucose phosphate
Glucose 6-phosphate + NADP+
6-phosphogluconate + NADPH + H+
Major function of the pentose phosphate pathway is
supplying the cell with NADPH which in turn carries
electrons to biosynthetic reactions