SEMESTER 1 (2012/2013)

BIOCHEMICAL ENGINEERING PTT 203

PN. NURUL AIN HARMIZA ABDULLAH

P A G E 1 3 3 . S H U L E R , M . L . A N D K A R G I . ( 2 0 0 2 ) . B I O P R O C E S S E N G I N E E R I N G : B A S I C C O N C E P T . 2 N D 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

H A L L P T R

Chapter 5 : MAJOR METABOLIC

PATHWAYS

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

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

Why we need to learn the metabolic pathways?

Starch smaller glucose

amylase

external internal

• 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.

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

Catabolism

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.

Which one is EXERGONIC and which one is ENDERGONIC ?

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)

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

Which Class is CATABOLISM and which is ANABOLISM?

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

It contains high-energy phosphate bonds.

The energy in ATP is obtained from the breakdown of foods.

ATP

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

Glycolysis

Where is this GLYCOLYSIS took place in a

cell?

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

• 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

What is NAD ?

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?

Citric acid cycle

Where is this CAC took place in a

cell?

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?

phosphofructokinase (active) phosphofructokinase (inactive)

ATP

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.

Glycolysis

The phosphorylation of fructose 6-phosphate is

highly exergonic and irreversible, and

phosphofructokinase, the enzyme that catalyzes it,

is the key enzyme in glycolysis.

Rate-limiting enzyme for glycolysis is phosphofructokinase

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, NO3-

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.