Glycolysis/EMP Pathway

Glycolysis/EMP

Glycolysis degrade a molecule of glucose in a series of enzyme-catalyzed reactions to give/yield 2 molecules of three-carbon(C3) compound pyruvate of lower free energy. Free energy released from glucose is conserved (use to synthesis) in the form of ATP and NADH.

·       Glycolysis reactions are take place in cytosol.

Major contributors:

1.     Gustav Embden

2.     Otto Meyerhof

3.     Jacob Parnas

Chemical Strategy of Glycolysis is:

1. Add phosphoryl groups to the glucose
2. Chemically convert phosphorylated intermediates into compounds with high phosphate group-transfer potentials.
3. Chemically couple the subsequent hydrolysis of reactive substances to ATP synthesis.

Stages of Glycolysis

Glycolysis has 10 steps which can dived into 2 phases.

• ·       Stage I (Reactions 1–5) - A preparatory stage - hexose glucose is phosphorylated and cleaved to yield 2 molecules of the triose glyceraldehyde-3-phosphate. This process utilizes 2 ATPs in a kind of energy investment 

• ·       Stage II (Reactions 6–10): Payoff phase -

The 2 molecules of glyceraldehyde-3-phosphate are oxidative converted to pyruvate, with generation of 4 ATPs & 2NADH.so, net ATP gain per glucose molecule in glycolysis is 2.

extra -

Energy Remaining in Pyruvate

Glycolysis releases only a small fraction of the total available energy of the glucose molecule; the two molecules of pyruvate formed by glycolysis still contain most of the chemical potential energy of glucose.

    

Importance of Phosphorylated Intermediates

Each of the 9 glycolytic intermediates between glucose and pyruvate is phosphorylated. The phosphoryl groups appear to have 3 functions -

1. Because the plasma membrane generally lacks transporters for phosphorylated sugars, the phosphorylated glycolytic intermediates cannot leave the cell. After the initial phosphorylation, no further energy is necessary to retain phosphorylated intermediates in the cell, although the large difference in their intracellular and extracellular concentrations 
2. Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. (extra - Energy released in the breakage of phosphoanhydride bonds (such as those in ATP) is partially conserved in the formation of phosphate esters such as glucose 6-phosphate. High-energy phosphate compounds formed in glycolysis (1,3-bisphosphoglycerate and phosphoenolpyruvate) donate phosphoryl groups to ADP to form ATP)
3. Binding energy resulting from the binding of phosphate groups to the active sites of enzymes lowers the activation energy and increases the specificity of the enzymatic reactions. (extra - The phosphate groups of ADP, ATP, and the glycolytic intermediates form complexes with Mg²⁺ and the substrate binding sites of many glycolytic enzymes are specific for these Mg²⁺ complexes. Most glycolytic enzymes require Mg²⁺ for activity)

Reaction 1 -Phosphorylation of Glucose

• 1^st Reaction of glycolysis is the transfer of a phosphoryl group from ATP to glucose to form glucose-6-phosphate (G6P) in a reaction catalyzed by hexokinase (HK)
• kinases are the enzymes that transfers phosphoryl groups between ATP and a metabolite
• Hexokinase, like many other kinases, requires Mg²⁺ for its activity, because the true substrate of the enzyme is not ATP^4- but the MgATP^2- complex. Mg²⁺ shields the negative charges of the phosphoryl groups in ATP, making the terminal phosphorus atom an easier target for nucleophilic attack by an -OH of glucose

Reaction 2 - Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate

• conversion of G6P to fructose-6-phosphate (F6P) by phosphoglucose isomerase (PGI; also called glucose-6-phosphate isomerase/ phosphohexose isomerase)
• This is the isomerization of an aldose to a ketose

Reaction 3 - Phosphorylation of Fructose 6-Phosphate to Fructose 1,6- Bisphosphate

• phosphofructokinase (PFK) catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate(FBP) [previously known as fructose-1,6-diphosphate (FDP)]
• PFK plays a central role in the control of glycolysis because it catalyzes one of the pathway’s rate-determining reactions

Reaction 4 - Cleavage of Fructose 1,6-Bisphosphate

• Aldolase catalyzes the cleavage of FBP to form the 2 trioses glyceraldehyde-3-phosphate (GAP)and dihydroxyacetone phosphate (DHAP)
• Aldol cleavage between C3 and C4 of FBP requires a carbonyl at C2 and a hydroxyl at C4.

Reaction 5- Interconversion of the Triose Phosphates

• DHAP and GAP are ketose–aldose isomers
• Triose phosphate isomerase (TIM or TPI) catalyzes this process

Reaction 6- Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Bisphosphoglycerate

• This involves the oxidation and phosphorylation of GAP to 1,3-bisphosphoglycerate by NAD⁺ and P_i as catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
• aldehyde oxidation, an exergonic reaction, drives the synthesis of the acyl phosphate group at C-1 1,3-bisphosphoglycerate.

Reaction 7- Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP

• phosphoglycerate kinase(PGK) transfers the high-energy phosphoryl group from the carboxyl group of 1,3-bisphosphoglycerate to ADP, forming ATP and 3- phosphoglycerate.

Reaction 8- Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

• phosphoglycerate mutase(PGM) catalyzes the transfer of phosphoryl group between C-2 and C-3 of glycerate which results in conversion of 3PG to 2-phosphoglycerate (2PG).(mutases catalyze the transfer of a functional group from one position to another on a molecule)

Reaction 9 - Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate

• 2PG is dehydrated to phosphoenolpyruvate (PEP)in a reaction catalyzed by enolase
• The enzyme forms a complex with a divalent cation such as Mg²⁺ before the substrate is bound

Reaction 10 - Transfer of the Phosphoryl Group from PEP to ADP

• Transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K⁺ and either Mg²⁺ or Mn²⁺
• This is a substrate-level phosphorylation

Overall reaction of glycolysis -

Glucose +2NAD⁺ +2ADP +2P_i →

2 pyruvate +2NADH +4H⁺ +2ATP +2H₂O

Regulation of Glycolysis

Enzyme	Inhibitors	Activators
Hexokinase	G-6-P
Phosphofructokinase	ATP, PEP, Citrate	ADP, AMP, Fructose 2,6-P
Pyruvate kinase	ATP

The Oxidizing Power of NAD⁺ Must Be Recycled

• NAD⁺ is the primary oxidizing agent of glycolysis
• The NADH produced by this process must be continually re-oxidized to keep the pathway supplied with NAD⁺

There are three common ways that this occurs -

1. Under anaerobic conditions in muscle, NAD⁺ is regenerated when NADH reduces pyruvate to lactate.
2. Under anaerobic conditions in yeast, pyruvate is decarboxylated to yield CO₂ and acetaldehyde and the latter is reduced by NADH to yield NAD⁺ and ethanol.
3. Under ATPs aerobic conditions, the mitochondrial oxidation of each NADH to NAD⁺ yields 2.5

Thus, in aerobic glycolysis, NADH may be thought of as a “high-energy” compound, whereas in anaerobic glycolysis its free energy of oxidation is dissipated as heat.

References - 

1. Lehninger Biochemistry
2. BIOCHEMISTRY by VOET . D & VOET J.G
3. Bacterial Metabolism by Gerhard Gottschal 
4. MICROBIAL PHYSIOLOGY by Albert G. Moat , John W. Foster & Michael P. Spector

Special thanks - 

Dr. Gagandeep Kaur

DNA microarray

DNA microarray

A DNA microarray also known as DNA chip or a Biochip.

•            Definition - DNA microarrays are solid supports, usually of glass, nylon or silicon, on which Collection of microscopic DNA spots (DNA probes) are immobilized as microdots in an organized grid fashion. Each spot of DNA, called a probe, represents a single gene.

It allows the measurement of the level of gene expression for every gene in genome.

Principle

The principle of DNA microarrays lies on the hybridization between the nucleotide. Using this technology, the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization.

Hybridization: The property of complementary nucleic acid sequences is to specifically pair

with each other by forming hydrogen bonds between complementary nucleotide base pairs.

Requirements - 

1. DNA chip
2. Target sample
3. Fluorescent dyes
4. Probes
5. Scanner
6. Enzymes

Steps -

1. Sample preparation
2. Purification (Isolate mRNA)
3. Reverse Transcription
4. Labeling
5. Hybridization
6. Scanning
7. Normalization and analysis.

Sample preparation - We’ll use two samples – cancerous human skin tissue & healthy human skin tissue.

Purification (Isolate mRNA) –

•         Extract the RNA from the samples. Using either a column, or a solvent such as phenol-chloroform.
•         isolate the mRNA from total RNA by using affinity column containing beads with Poly-T tails to bind the mRNA. Rinse with buffer to release the mRNA from the beads.

Reverse Transcription & Labeling

•         Prepare cDNA by reverse transcription by using cyanine 3 (fluoresces green) labeled nucleotides for the healthy cell’s mRNA template and cyanine 5 (fluoresces red) labeled nucleotides for cancerous cell mRNA template. then finally degrade the mRNA.

Hybridization

•        Hybridize the labeled cDNA with DNA probes immobilized on microarray plate.
•        Computer data base having which gene contain in each spot.
•     after hybridizing wash to remove unhybridized cDNA.

Scanning

•         scanner consists with laser, a computer, and a camera.
•         The camera records the images produced when the laser scans the plate. The computer helps in view results & the store data. 

Normalization and analysis

• Green spots → cDNA from healthy tissue hybridized to the target DNA
• Red spots → cDNA from diseased/Cancer tissue hybridized to the target DNA (this is the interest result in disease/cancer diagnosis)
• Yellow spots → both healthy & diseased tissue cDNA hybridized equally to the target DNA (still the gene can carry out is function.
• Black spots → neither healthy or diseased tissue cDNA hybridized to the target DNA   

There are 2 types of DNA Chips/Microarrays - 

1. cDNA based microarray
2. Oligonucleotide based microarray

Applications-

1. Gene expression profiling –  In different cells/tissues, Under different environmental or chemical stimuli, In disease state versus healthy.
2. Discovery of drugs
3. Diagnostics – Microbial identification, microbial genotyping, Antibody detection, Cancer detection
4. Toxicological research (Toxicogenomics)
5. Pharmacogenomics - Individualized medicine.

For more information checkout this video -

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Recombinant insulin Production

Recombinant insulin Production

Insulin, synthesized by the β-cells of the islets of Langerhans in the pancreas, controls the level of glucose in the blood.

An insulin deficiency manifests itself as diabetes mellitus, a complex of symptoms which may lead to death if untreated. 

Insulin used in this treatment was originally obtained from the pancreas of pigs and cows.

Problems of animal-derived Insulin

1. Side effects in some patients
2. Purification procedures are difficult
3. Potentially dangerous contaminants cannot always be completely removed

Features that facilitate its production by recombinant DNA techniques - 

1. Human protein is not modified after translation by the addition of sugar molecules
2. Size of the molecule is small, comprising two polypeptides, one of 21 amino acids (the A chain) and one of 30 amino acids (B chain)

Preproinsulin precursor which contains the A and B segments linked by a third chain (C) and preceded by a leader sequence.

Method -

1. The leader sequence is removed after translation and the C chain excised, leaving the A and B polypeptides linked to each other by two disulphide bonds.
2. Then the artificial gene was ligated to a lacZ′ reading frame present in a pBR322-type vector of E. coli.
3. The insulin genes were expressed as fusion proteins, consisting of the first few amino acids of β-galactosidase followed by the A or B polypeptides which were separated by a methionine residue.
4. Then insulin polypeptides cleaved from the β-galactosidase segments by treatment with cyanogen bromide.
5. Then purify the A chains & B chains then finally carry-out disulphide bond formation

Citric Acid & vinegar Production

Citric Acid Production

Microbes involved

Aspergillus niger (mainly)

Asp. wenti

Candida quillermondi

Candida lipolytica

Candida oleiphila.

Basis of the Production of Citric Acid

Citric acid is an intermediate in the citric acid cycle (TCA). The acid can accumulate by one of the following methods –

a.     By mutation – giving rise to mutant organisms which may only use part of a metabolic pathway, or regulatory mutants; that is using a mutant lacking an enzyme of the cycle.

b.     altering the environmental conditions - temperature, pH, medium composition

                           i.          isocitrate dehydrogenase, are inhibited by anaerobiosis, hence limited aeration is done on the fermentation so as to increase the yield of citric acid.

                         ii.          Low pH

                       iii.          Addition of small amount of citric acid

Conditions & Requirements

1.     Media –

C source –

            N source -  Organic & Inorganic (ammonium nitrate)

Salts & Minerals

Growth factors

Water

2.      Temperature - 30⁰ C

3.      pH – 3.5 (HCl is added to adjust the medium to low pH)

4.      Time – 5-14 days

5.      Trace elements -  Zn, Mn, Fe, Cu. use to initiate the reaction

6.      KH₂PO₄, MgSO₄

7.     As high aeration is deleterious to citric acid production

8.     mechanical agitation is not necessary and air may be bubbled through

Production

Fermentation can be either surface or submerged.

Surface fermentation:

Mainly do in Japan by using rice bran

culture-  Aspergillus niger

Temp. pH, Time, media – same

aeration - limited

Submerged fermentation:

culture-  Aspergillus niger (mainly)

Temp. pH, Time, media – same

2 salts are added.  MgSO₄, 7H₂O – 1%

                              KH₂PO₄ - 0.05-2%

Copper is used up to 500 ppm as an antagonist of the enzyme aconitase which requires iron

If beet molasses used as the substrate – Ferro-cyanide is added to reduce Fe percentage.

Harvest/ Recovery/Downstream process/Extraction (draw a flow chart)

The broth is filtered until clear & extract citric acid.

Add Ca(OH)₂ to precipitate Calcium citrate

Wash the precipitate & add Dil. H₂SO₄ ; it results CaSO₄ + citric acid

then, Dried to form crystals of citric acid.

Powder & package

Uses-

1.     Food industry

a.      as food acidulant in the production of jams, sweets, and soft drinks.

b.     as an artificial flavoring

c.      Production of processed cheese (Sodium citrate)

2.     medicine and pharmaceutical industry

a.      blood transfusion & prevention of blood clotting. (Sodium citrate)

b.     as a source of energy

3.     cosmetic industry

a.      In astringent lotions (e.g.- aftershave lotions)

b.     In hair sprays

4.     Other

a.      as a chelating agent; it chelates Fe³⁺ & help in oil recovery

b.     Production of Detergents.

Acetic acid/ Vinegar Production

Vinegar is a product resulting from the conversion of alcohol to acetic acid by acetic acid bacteria, Acetobacter spp

Types of Vinegar

1.     Cider vinegar, apple vinegar - from fermented apple justice

2.     Wine vinegar, grape vinegar – from Fermented grape juice

3.     Malt vinegar - from fermented barley malt

4.     Spirit vinegar - from distilled alcohol

5.     Flavored vinegar

Microbes involved

Acetobacter aceti

Gluconobacter oxydans

bacteria should have following characteristics –

1.     tolerate high concentrations of acetic acid

2.     require small amounts of nutrient

3.     not over oxidize the acetic acid formed

4.     should have high yield

.

CH₃CH₂OH + (O)    →                             CH₃CHO + H₂O

Ethyl alcohol oxygen                                                 Acetaldedyde Water

CH₃CHO + H₂O          →                             CH₃CH(OH)₂

Hydrated acetaldehyde

CH₃CH(OH)₂ + (O)       →                          CH₃COOH + H₂O

                                       Dehydrogenase              Acetic acid

1 gm of alcohol →yield 1.304 gm of acetic

Production

The 3 methods used for the production of vinegar

1.     Orleans Method (slow method)

2.     Trickling (or quick) Method

3.      Submerged Fermentation

Orleans Method (slow method) – now days not use (see pic in written book)

·       wine left in open vats/tubs became converted to vinegar by acetic acid bacteria entering it from the atmosphere.

·       wine was put in wooden vessels and left in the open & Add small amount of vinegar to initiate fermentation as an inoculum of acetic acid bacteria

·       then, a thick film (mother liquor film) of acetic acid bacteria formed on the wine and converted it in to vinegar in about 5 weeks

Limitations

1.     Slow process

2.     less efficient, yielding 75-85%

3.     contamination

The Trickling Generators (Quick) Method (see pic in written book)

·       The vessel is made out of wood or stainless steel.

·       broth + culture add only up to 4/5 of total volume.

·       alcohol-acetic acid mixture spray at the top.

·       wood pieces of the broth – provide surface for biofilm formation

·       cooling water jacket is used to regulate the temperature between 29°C - 35°C. this is determined by thermometers placed in vessel.

·       ethyl alcohol level must be maintained; not fall below 0.3-0.5%

·       Final acidity of the vinegar is about 12%

·       Have 80% of efficiency.

Advantages

1.     High yield

2.     Contamination decreased.

Submerged Fermentation

aeration is crucial due shortage of oxygen because of the highly acid conditions of submerged production

Can be carried out by –

a.      Fringes’ acetator

b.     The tower fermenter

Fringes’ acetator

·       It consists of a stainless steel tank fitted with internal cooling coils and a high speed agitator fitted through the bottom.

·       Temperature -  30°C

·       Air is sucked at supplied to the fermenter

·       Foaming is interrupted with an automatic foam breaker

·       operated batch wise, and 35hrs batch cycle produce 12% vinegar

Advantages

1.     High yield

2.     smaller space required.

3.     Automatic system; so easy in operation

4.     Can produce multiple types of vinegar

The tower fermenter

·       developed in the UK

·       The fermenter is 2 feet in diameter, 20 feet tall in the tubular section; expansion chamber 4 feet in diameter and 6 feet high.

·       working volume -  3,000 liters

Harvest/ Recovery

1.     Clarification by Filtration

             Submerged method much turbid than tricking method and so filtration is much

             important in Submerged method. Kieselguhr use as a filter aid in filtration process

2.     pasteurized at 60-65°C for 30 minutes

3.     concentrated by freezing