Matabolic Stoichiometry and Matabolic Stoichiometry and Energetics in MicroorganismsEnergetics in Microorganisms

Dr. A.K.M. Shafiqul IslamDr. A.K.M. Shafiqul Islam

MetabolismMetabolism

A living cell is a complex chemical reactor in which A living cell is a complex chemical reactor in which more than 1000 independent enzyme-catalyzed more than 1000 independent enzyme-catalyzed reaction occursreaction occurs– The total of all chemical reaction activities which occur The total of all chemical reaction activities which occur

in the cell is called metabolism.in the cell is called metabolism.

The metabolic reaction tend to be organized into The metabolic reaction tend to be organized into sequences called metabolic pathways which sequences called metabolic pathways which connect one reaction with anotherconnect one reaction with another

Important CoenzymesImportant Coenzymes

NAD+NAD+ NADP+NADP+ FADFAD Coenzyme ACoenzyme A

A cell produces order from its disorderly A cell produces order from its disorderly surrounding thingssurrounding things

Energy from the environment is used to drive the Energy from the environment is used to drive the metabolic processmetabolic process

In bioprocess engineering, the energy exchanges In bioprocess engineering, the energy exchanges helps explain the major distinction between cell helps explain the major distinction between cell function in the presence and absence of oxygenfunction in the presence and absence of oxygen

Types of MetabolismTypes of Metabolism

Three types of metabolismThree types of metabolism– AerobicAerobic

Use free oxygen Use free oxygen

– Anaerobic Anaerobic Do not use free oxygenDo not use free oxygen

– facultative anaerobes facultative anaerobes A third class of cells can grow in either environment and A third class of cells can grow in either environment and

known as facultative anaerobes. Yeast is a familiar known as facultative anaerobes. Yeast is a familiar example of this metabolic variety example of this metabolic variety

Two different kinds of energy are tapped by Two different kinds of energy are tapped by inhabitants of microbial worldinhabitants of microbial world– Light Light

Organism which relay on light are called Organism which relay on light are called phototrophsphototrophs

– Chemical Chemical While While chemotrophschemotrophs extract energy by breaking down extract energy by breaking down

certain nutrients.certain nutrients.

Further subdivision of chemotrophs is Further subdivision of chemotrophs is possiblepossible– LithotrophsLithotrophs

Oxidize organic materialsOxidize organic materials

– OrganotrophsOrganotrophs Employ organic nutrients for energy productionEmploy organic nutrients for energy production

The energy obtained from the environment is stored and The energy obtained from the environment is stored and shuttled in the high-energy intermediates such as ATP. shuttled in the high-energy intermediates such as ATP. Cell use this energy to perform three types of work:Cell use this energy to perform three types of work:

– chemical synthesis of large or complex moleculeschemical synthesis of large or complex molecules

– transportation of ionic or neutral substances into or out of the cell transportation of ionic or neutral substances into or out of the cell or its internal organcellsor its internal organcells

– mechanical work required for cell division and motionmechanical work required for cell division and motion

All these processes are (by themselves) All these processes are (by themselves) nonspontaneous and result in an increase of free energy nonspontaneous and result in an increase of free energy of the cell. They occur when simultaneously couple to of the cell. They occur when simultaneously couple to another process which has a negative free-energy another process which has a negative free-energy change of greater magnitude.change of greater magnitude.

In order to grow and reproduce, cells must In order to grow and reproduce, cells must ingest the raw materials necessary to ingest the raw materials necessary to manufacture membrane, protein, walls, manufacture membrane, protein, walls, chromosomes and other componentschromosomes and other components

Four major requirements are evident: Four major requirements are evident: – carbon, nitrogen, sulfur and phosphoruscarbon, nitrogen, sulfur and phosphorus

Reactions within the cell have been subdivided Reactions within the cell have been subdivided into three classes:into three classes:

– degradation of nutrientsdegradation of nutrients– biosynthesis of small moleculesbiosynthesis of small molecules– biosynthesis of large macromoleculesbiosynthesis of large macromolecules

Each reactions are catalyzed by an enzyme. The Each reactions are catalyzed by an enzyme. The enzyme serve the essential function of enzyme serve the essential function of determining which reaction occur and their relative determining which reaction occur and their relative ratesrates

Thermodynamic Principles Thermodynamic Principles

To get an idea of whether a certain reaction in the To get an idea of whether a certain reaction in the cell will run forward or backword, we will use a cell will run forward or backword, we will use a number of approximation since full analysis of number of approximation since full analysis of metabolic network is not practical. First we metabolic network is not practical. First we consider the free-energy change of a chemical consider the free-energy change of a chemical reactionreaction

(1)(1)

We can writeWe can write

(2)(2)

DCBA

ba

dcRTGG ln'0

In a closed system, the reaction will proceed left to right if In a closed system, the reaction will proceed left to right if and only if and only if GG‘ is negative. Accordingly, ‘ is negative. Accordingly, GG‘ is zero at ‘ is zero at equilibrium give the following realtionshipequilibrium give the following realtionship

(3)(3)

wherewhere

If water or HIf water or H++ are involve in the reaction, their are involve in the reaction, their concentrations do not enter into the calculation of the right concentrations do not enter into the calculation of the right hand side (4). The value already includes the water and Hhand side (4). The value already includes the water and H++ concentration (for pH 7)concentration (for pH 7)

eqKRTG ln'0

eqeq

eqeqeq ba

dcK (4)

Consider the reaction between two isomers in the Consider the reaction between two isomers in the Embden-Mayerhof pathway for glucose breakdownEmbden-Mayerhof pathway for glucose breakdown

Where P denotes phosphate. Because of the negative free Where P denotes phosphate. Because of the negative free energy change, equilibrium favors the dihydroxyacetone by energy change, equilibrium favors the dihydroxyacetone by a 22:1 ratio.a 22:1 ratio.

CHO

CHOH

CH2O P

Glyceraldehyde3-phosphate

CH2OH

C

CH2O P

Dihydroxyacetone-P

O G0’= -1830 cal/mol

Many biological reaction and energy conversion process Many biological reaction and energy conversion process involve oxidation-reduction reaction such asinvolve oxidation-reduction reaction such as

This type of reaction is described using the standard This type of reaction is described using the standard potential changepotential change

oxredredox BABA

0

BB0

AA0

redBoxredoxEEΔE

where is the standard half-cell potential for the half reaction

0Aox

E redA

redox A2eA

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

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

Where Where nn is the number of electrons transferred is the number of electrons transferred and and FF is equal to 23.062 kcal/V molis equal to 23.062 kcal/V mol

2H2e2H 0)(pH 0.000VE0

EnG F

Metabolic Reaction Coupling:Metabolic Reaction Coupling:ATP and NADATP and NAD

ATPATP

Energy is released as food is oxidizedEnergy is released as food is oxidized Used to form ATP from ADP and PUsed to form ATP from ADP and Pii

ADP + PADP + Pii + + EnergyEnergy ATPATP

In cells, energy is provided by the hydrolysis In cells, energy is provided by the hydrolysis

of ATPof ATP

ATPATP ADP + P ADP + Pii + + EnergyEnergy

The enzymatic hydrolysis of ATP to yield ADP and The enzymatic hydrolysis of ATP to yield ADP and inorganic phosphate has a large negative free-inorganic phosphate has a large negative free-energy changeenergy change

Where Where PPii indicate inorganic phosphate indicate inorganic phosphate

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

By reversing the reaction and adding the By reversing the reaction and adding the phosphate to ADP, free energy can be stored for phosphate to ADP, free energy can be stored for late use late use

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

Embden-Meyerhof-Parnas pathway serves to illustrate Embden-Meyerhof-Parnas pathway serves to illustrate the concept of a the concept of a common chemical intermediatecommon chemical intermediate

1.1. Oxidation of aldehyde to carboxylic acidOxidation of aldehyde to carboxylic acid

2.2. Same reactions, coupled to ATP generation (glucose Same reactions, coupled to ATP generation (glucose oxidation)oxidation)

HRCOOH2OHRCHO 2

kcal/mol7G01

43-24 ATPRCOOH2ADPHPORCHO

kcal/mol0G02

3.3. Reaction 2 and 1 yieldReaction 2 and 1 yield

OHATPHPOADP 24-2

43 H

kcal/mol7G03

ExampleExample

P OO

O-

-O C C C HH

H

H

OH

O+ HPO4

=

P OO

O-

-O C C C OH

H

H

OH

O2H H2O ++ P O-

O

O-

P O

O

O-

-O C C C O

H

H

H

OH

O

P O-

O

O-

RCOO- + ATP4-

ADP3-+

Thus glucose metabolism is the process at which cell Thus glucose metabolism is the process at which cell generates the ATP needed for endergonic processgenerates the ATP needed for endergonic process

This generation is accomplished by the conversion of a This generation is accomplished by the conversion of a partially metabolized nutrient into a high-energy partially metabolized nutrient into a high-energy phosphorylated intermediate, which then donates a phosphorylated intermediate, which then donates a phosphate to ADP via an enzyme-catalyzed reactionphosphate to ADP via an enzyme-catalyzed reaction

The phosphorylation of various The phosphorylation of various compounds serves several functionscompounds serves several functions

It provides a useful means of storing considerable fractions of It provides a useful means of storing considerable fractions of free energy of fuel oxidation. Free energies of hydrolysis of free energy of fuel oxidation. Free energies of hydrolysis of several called phosphate donors are greater than several called phosphate donors are greater than GG00’’ for for ATP hydrolysis.ATP hydrolysis.Example, phosphoenolpyruvate Example, phosphoenolpyruvate GG00’’ = -14.8 kcal mol = -14.8 kcal mol-1-1..

1,3-diphosphoglycerate 1,3-diphosphoglycerate GG00’’ = -11.8 kcal mol = -11.8 kcal mol-1-1

Hydrolysis of this compounds can be used to drive ADP Hydrolysis of this compounds can be used to drive ADP phosphorylationphosphorylation

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

Example, glucose-6-phosphate Example, glucose-6-phosphate GG00’’ = -3.3 kcal mol = -3.3 kcal mol-1-1

glycerol-1-phosphate glycerol-1-phosphate GG00’’ = -2.2 kcal mol = -2.2 kcal mol-1-1

Highly ionized organic substances are virtually unable to Highly ionized organic substances are virtually unable to permeate the cell’s plasma membranes. The charged permeate the cell’s plasma membranes. The charged phosphorylated compounds which serves as metabolic phosphorylated compounds which serves as metabolic intermediates may therefore be contained within the cell. intermediates may therefore be contained within the cell. Thus maximum amounts of energy and chemical raw Thus maximum amounts of energy and chemical raw materials can be extracted from a nutrient.materials can be extracted from a nutrient.

Oxidation reduction: Oxidation reduction: Coupling via NADCoupling via NAD

Oxidation-reduction reactions are conducted biologically Oxidation-reduction reactions are conducted biologically

and the connection between these mechanisms and ATP and the connection between these mechanisms and ATP metabolism.metabolism.

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

and that addition of electron is reduction of a compound.and that addition of electron is reduction of a compound.

When an organic compound is oxidized biologically, it When an organic compound is oxidized biologically, it usually loses electrons in the form of hydrogen atomsusually loses electrons in the form of hydrogen atoms

similarly, hydrogenation is the usual way of adding electronsimilarly, hydrogenation is the usual way of adding electron

CH3

C

COOH

O

Pyruvicacid

Lacticacid

CH3

HC

COOH

OH+ 2H (reductionof pyruvic acid)

+ 2H (oxidationof lactic acid)

N

NN

N

NH2

O

HOH

HHHH

O

P

O

P

O

O

O O

HOH

HHHH

N

CONH2

HO

OH

Nicotinamide adenine dinucleotide (NAD)

Pairs of hydrogen atoms freed during oxidation or required Pairs of hydrogen atoms freed during oxidation or required in reductions are carried by nucleotide derivatives, in reductions are carried by nucleotide derivatives, especially nicotinamide adenine dinucleotide (NAD) and its especially nicotinamide adenine dinucleotide (NAD) and its phosphorylated form of NADP.phosphorylated form of NADP.

HCC

HCN

CH

C NH2

O

R

H

HCC

HCN

CH

C NH2

O

R

HH

- 2H (oxidation)

+ 2H (reduction)

NADH NAD+

Reduction form Oxidation form

NAD serves two major functionsNAD serves two major functions

1.1. Analog to one of ATP’s job – Analog to one of ATP’s job –

reducing power made available during breakdown of reducing power made available during breakdown of nutrient is carried to biosynthetic reaction. The nutrient is carried to biosynthetic reaction. The reducing power is used for the construction of cell reducing power is used for the construction of cell components.components.

When a metabolite is oxidized, NADWhen a metabolite is oxidized, NAD++ accepts two electrons accepts two electrons plus a hydrogen ion (Hplus a hydrogen ion (H++) and NADH results.) and NADH results.

NADH then carries energy to cell for other usesNADH then carries energy to cell for other uses

NAD and related pyridine nucleotide compounds carrying NAD and related pyridine nucleotide compounds carrying hydrogen also participate in ATP formation in aerobic hydrogen also participate in ATP formation in aerobic metabolism. The hydrogen atoms in NADH are combined metabolism. The hydrogen atoms in NADH are combined with oxygen in a cascade of reactions known as the with oxygen in a cascade of reactions known as the respiratory chain. The energy released in this oxidation is respiratory chain. The energy released in this oxidation is sufficient to form three molecule of ATP from ADP.sufficient to form three molecule of ATP from ADP.

All the biological systems, e.g., anaerobic, aerobic, or All the biological systems, e.g., anaerobic, aerobic, or photosynthetic metabolism, utilize ATP as central means of photosynthetic metabolism, utilize ATP as central means of accumulating oxidative or radiant energy for driving the accumulating oxidative or radiant energy for driving the endergonic processes of the cell.endergonic processes of the cell.

CARBON CATABOLISMCARBON CATABOLISM

Breakdown of nutrients to obtain energy is called Breakdown of nutrients to obtain energy is called catabolism.catabolism.

Fermentation of carbohydrates, e.g., glucose, are under Fermentation of carbohydrates, e.g., glucose, are under this category. this category.

The are at least seven glucose fermentation pathways and The are at least seven glucose fermentation pathways and the particular one used and the end products produced the particular one used and the end products produced depend on the microorganism involveddepend on the microorganism involved

Embden-Meyerhof-Parnas Pathway (EMP)Embden-Meyerhof-Parnas Pathway (EMP)

Embden-Meyerhof-Parnas Pathway involved in ten enzyme Embden-Meyerhof-Parnas Pathway involved in ten enzyme catalyzed steps which start with glucose and end with catalyzed steps which start with glucose and end with pyruvate.pyruvate.

The EMP steps involve isomerization, ring splitting, or The EMP steps involve isomerization, ring splitting, or transfer of a small group such as hydrogen or phosphate.transfer of a small group such as hydrogen or phosphate.

Two moles of pyruvate are produced per mole of glucose Two moles of pyruvate are produced per mole of glucose passing through the pathway.passing through the pathway.

ATP hydrolysis coupled with two reactions and each ATP hydrolysis coupled with two reactions and each reaction involve sufficiently negative free negative energies reaction involve sufficiently negative free negative energies to drive ADP phosphorylation. to drive ADP phosphorylation.

OH

OH

H

OH

H

OHH

OH

CH2OH

HHexokinase

ATP ADPOH

OH

H

OH

H

OHH

OH

CH2OPO32-

H

OH

CH2OH

H

CH2OPO32-

OH H

H OH

OPhosphohexoisomerase

OH

CH2OPO32-

H

CH2OPO32-

OH H

H OH

O

Phospho-f ructokinase

ATP

ADP

CH2OPO32-

C

CH2OH

O

HC

HCOH

CH2OPO32-

O

Glucose Glucose 6-phosphate Fructose 6-phosphate

Fructose 1,6-diphosphateDihydroxyacetone phosphate

Glyceraldehyde 3-phosphate

Triose isomerase Aldolase

HC

HCOH

CH2OPO32-

O

Glyceraldehyde 6-phosphate

CH2OPO32-

HCOH

C OPO32-

O

CH2OPO32-

HCOH

COO-

3-Phosphoglycerate

Glyceraldehyde 3-phosphatedihydrogenase

NAD+ NADH

3-phosphoglyceratekinase

ATPADP

1,3-Diphosphoglycerate

CH2OH

HCOPO32-

COO-

2-Phosphoglycerate

Phosphoglyceramutase

CH2

C

COO-

O PO32-

CH3

C

COO-

OEnolase

H2O

PhosphoenolpyruvatePyruvate

Pyruvate kinase

ATPADP

CC66HH1212OO66 + 2 P + 2 Pii + 2 ADP + 2 NAD + 2 ADP + 2 NAD++

Stored chemical energy and reducing power result from Stored chemical energy and reducing power result from overall pathway. This is called overall pathway. This is called substrate-level pathwaysubstrate-level pathway

In muscle cell and lactic acid bacteria, the reactions of the In muscle cell and lactic acid bacteria, the reactions of the EMP are followed by single stepEMP are followed by single step

The overall reaction sequence from glucose to lactic acid is The overall reaction sequence from glucose to lactic acid is called called glycolysisglycolysis

CC33HH44OO33 + NADH + H + NADH + H++ CC33HH66OO33 + NAD + NAD++

2 C2 C33HH44OO33 + 2 ATP + 2 (NADH + H + 2 ATP + 2 (NADH + H++))

Free-energy change for overall glycolysis reactionFree-energy change for overall glycolysis reaction

With corresponding quantity for the glucose breakdown With corresponding quantity for the glucose breakdown alonealone

A total free-energy of 14.6 kcal or 7.3 kcal for each mole of 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 ATP generated has been conserved by the pathway as high energy phosphate compounds.high energy phosphate compounds.

Glucose + 2 PGlucose + 2 Pii + 2 ADP + 2 ADP 2 lactose + 2 ATP + 2 H 2 lactose + 2 ATP + 2 H22OO

G0’ = -32,400 cal/mol

Glucose Glucose 2 lactose 2 lactose

G0’ = -47,000 cal/mol

The breakdown of carbohydrates to release The breakdown of carbohydrates to release energyenergy– GlycolysisGlycolysis– Krebs cycleKrebs cycle– Electron transport chainElectron transport chain

Carbohydrate CatabolismCarbohydrate Catabolism

Other Carbohydrate Catabolic Other Carbohydrate Catabolic PathwaysPathways

The pentose phosphate cycle or pathway begins by The pentose phosphate cycle or pathway begins by oxidizing glucose phosphateoxidizing glucose phosphate

Major function of the pentose phosphate pathway is Major function of the pentose phosphate pathway is supplying the cell with NADPH which in turn carries supplying the cell with NADPH which in turn carries electrons to biosynthetic reactionselectrons to biosynthetic reactions

Glucose 6-phosphate + NADPGlucose 6-phosphate + NADP++

6-phosphogluconate + NADPH + H6-phosphogluconate + NADPH + H++