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Part twoprinciple of biochemistry

Metabolism and biological energy2-carbohydrate Metabolism 2-Tri-carboxylic Acid cycle

3-Electron Transport System

Course code: HFB324

Credit hours: 3 hours

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Tricarboxylic acid cycle

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• lactate: a 3-carbon compound produced from pyruvate during anaerobic metabolism

• oxaloacetate: a carbohydrate intermediate of the TCA cycle.

Oxidative phosphorylation is the process that conserves the energy of the ETC by phosphorylation of ADP to ATP

The chemiosmotic coupling theory explains how oxidative phosphorylation links the ETC and ATP synthesis

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• Cytochromes are, in general, membrane-bound (i.e. inner mitochondrial memberane) hemoproteins containing heme groups and are primarily responsible for the generation of ATP via electron transport

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2-Tri-carboxylic Acid cycle Citric Acid Cycle, Krebs Cycle

2nd phase of cellular respiration

• *kerb's cycle is a series of reactions in the Mitochondria that bring about the catabolism of acetyl residues, liberating hydrogen equivalent (2H) which on oxidation lead to the release of most of the free energy of tissue fuels.

• the acetyl residues are in the form of acetyl Co-enzyme A (active acetate).

• *reducing equivalent (electrons) are oxidized by respiratory chain with release of ATP.

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• It is the final pathway for oxidation of glucose, lipids and protein for the generation of ATP.

• It catalyzed the combination of their common metabolite----acetyl Co-enzyme A with oxaloacetate to form citrate by series of dehydrogenation and decarboxylation reaction ,

• citrate or citric acid is degraded releasing reducing equivalent (energy in the form of H molecules) and 2 carbon dioxide and regenerating oxaloacetate.

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The beginning of the cycle

• *lactic acid is oxidized to pyruvate and the pyruvate is oxidized by specific enzyme to acetyl-Co enzyme A.

• *acetyl-Co enzyme A (2C) is combined with another acid known as oxaloacetate (4C) to yield citric acid (6C).

• *one molecule of acetyl Co-enzyme A is oxidized to CO2 +H2O in each cycle.

• *the oxaloacetate regenerated react with another molecule of acetyl Co-enzyme A and the cycle is repeated

• *many specific enzymes enter in this reaction mainly Thiamin Pyrophosphatase (TPP)

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Citric acid cycle has two functions

• 1-function in anabolism and catabolism of carbohydrates, fatty acids and amino acids

• 2-provides intermediates for synthesis of compound required for the body functioning

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Location of citric acid cycle

• Located in the mitochondrial matrix• Mitochondrial membrane facilitates the

transfer of reducing equivalent H to the adjacent enzymes of respiratory chain

Mitochondria structure:1) inner membrane 2) outer membrane 3) cristae 4) Matrix

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3 stages of The Krebs Cycle

• 1. Acetyl CoA (2 C) binds a four carbon molecule (oxaloacetate) producing a six carbon molecule (citrate).

• 2. Two carbons are removed as carbon dioxide.

• 3. The four carbon starting material is regenerated.• The Krebs Cycle generates ATP and many energized

electrons (in the form of FADH2 and NADH) for the electron transport chain.

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Reactions of the TCA Cycleref. 1996–2012 themedicalbiochemistrypage.org, LLC | info @

themedicalbiochemistrypage.org

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1. Citrate synthase (synthesis of citric acid) The citric acid cycle begins when Coenzyme A

transfers its 2-carbon acetyl group to the 4-carbon compound oxaloacetate to form the 6-carbon

molecule citrate

Acetyl CoA and oxaloacetic acid condense to form citric acid. The acetyl group CH3COO is transferred from CoA to oxaloacetic acid at the ketone carbon, which is then changed to an alcohol.

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• The beginning step of the citric acid cycle occurs when;-

• a four carbon compound (oxaloacetic acid) condenses with acetyl CoA (2 carbons) to form citric acid (6 carbons)

• the starting point for the citric acid cycle.

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Step 2 aconitaseisomerization of the position of the -OH group on citric acid.

This first step is a dehydration of an alcohol to make an alkene The citrate is rearranged to form an isomeric form isocitrate

The citrate is rearranged into its isomer, isocitrate by the enzyme aconitase.

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3. Isocitrate DehydrogenaseOxidative decarboxylation of isocitrate to yield a -ketoglutarate The 6-carbon isocitrate is oxidized and a molecule of carbon dioxide is removed producing the 5-carbon molecule alpha-

ketoglutarate. During this oxidation, NAD+ is reduced to NADH + H+

First oxidative decarboxylation

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• This is the first step where a carbon group is lost as carbon dioxide in a decarboxylation reaction (oxidation reaction)

• The electron reduces NAD+ to NADH,• the proton is released as an H+ ion.

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4 -Ketoglutarate Dehydrogenase complex Alpha-ketoglutarate is oxidized, carbon dioxide is removed, and coenzyme A is added to form the 4-

carbon compound succinyl-CoA. During this oxidation, NAD+ is reduced to NADH + H+

high energy thioester succinyl-Co-A

A second oxidative decarboxylation

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• Second stage where NADH and the second CO2 are formed (A second oxidative decarboxylation)

Ketoglutarate Dehydrogenase complex need coenzymes (TPP, NAD, FAD and Co-A)

• Result of reaction is a high energy thioester succinyl-Co-A

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5. succinyl-CoA synthase (succinate thiokinase) CoA is removed from succinyl-CoA to produce succinate. The

energy released is used to make guanosine triphosphate (GTP) from guanosine diphosphate (GDP) and Pi by substrate-level

phosphorylation GTP can then be used to make ATP

substrate-level phosphorylation GTP can be used to make ATP

Succinic acid, a 4 carbon acid, is the product of this reaction(the beginning of the cycle).

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• The energy conserved from previous step in the succinyl-C A as the thioester bond is released in the form of ATP

• This is the only reaction where ATP is released at the substrate level

• The hydrolysis of the thioester bond (exothermic) is coupled with the formation of guanosine triphosphate first but is further coupled with the ADP to make ATP).

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6. Succinate dehydrogenase (flavoprotein)Succinate is oxidized to fumarate. During this

oxidation, two electrons and two protons produced are transferred to FAD, which becomes FADH2.

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• Succinic acid is degraded further to fumarate (4C) by the flavoprotein enzyme succinate dehydrogenase

• succinate dehydrogenase the only enzyme bound to inner surface of inner mitochondrial membrane

• The reaction produced FADH2

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7 Fumarase (fumarate hydratase)Water is added to fumarate to form malate (malic

acid)

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• In step 7 by the action of Fumarase water is added to fumarate to form malate (malic acid) this is a Hydration reaction to form an alcohol from alkene functional group

• This is a simple hydration reaction of an alkene (C to C=C) fumarate to form an alcohol (-OH is bound to a C atom) malate.

• Malate is freely permeable to mitochondrial membrane where then converted to oxaloacetate

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8 malate dehydrogenase. Malate is oxidized to produce oxaloacetate, the

starting compound of the citric acid cycle. During this oxidation, NAD+ is reduced to NADH + H+

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• Oxidation reaction of malate by the action of enzyme malate dehydrogenase

• This is the final reaction in the citric acid cycle. The reaction is the oxidation of an alcohol to a ketone to make oxaloacetic acid.

• The coenzyme NAD+ causes the transfer of two hydrogens and 2 electrons to NADH + H+.

• This is a final entry point into the electron transport chain (substrate level).

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Final products of citric acid cycle

• 2 acetyl groups + 6 NAD+ + 2 FAD + 2 ADP + 2 Pi

• forms;

• 4 CO2

• + 6 NADH• + 6 H+

• + 2 FADH2

• + 2 ATP

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3-Electron transport chainThe electron transport chain is third and final common pathway in aerobic cellular respiration to generate ATP.

• In this pathway electrons (reducing equivalents H+) are transferred to oxygen

• electrons transport between electron donor (NADH) and electron acceptor (O2).

• Passage of electrons between donor and acceptor releases energy This result is the formation of electrochemical proton gradient which used to generate ATP

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Mechanism of the chain

• Chemiosmotic theory• According to the theory, the transfer of electrons

down an electron transport system through a series of oxidation-reduction reactions releases energy.

• This energy allows certain carriers in the chain to transport hydrogen ions (H+ or protons) across a membrane

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• electrochemical gradient or potential difference across the membrane demonstrate the concentration of hydrogen ions on one side of the membrane

• One side of the membrane is positive (protons accumulate)

the other side is negative this lead to held the membrane to its energized state (proton motive force)

• The NADH + H+ and FADH2 carry protons and electrons to the electron transport chain to generate additional ATP by oxidative phosphorylation

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• the actions of the chain is carried on by highly organized oxidation-reduction enzymes, coenzymes and electron carrier cytochromes

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The purpose of the electron transport chain

• 1) to pass along 2H+ ions and 2e- to react with oxygen;

2) to conserve energy by forming three ATP's; and

3) to regenerate the coenzymes back to their original form as oxidizing agents

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Location of Electron transport chain

• This chain is located in the inner mitochondrial membrane of cell, protons are transported from the matrix of the mitochondria across the inner mitochondrial membrane to the intermembrane space located between the inner and outer mitochondrial membranes

Mitochondria structure:1) inner membrane 2) outer membrane 3) cristae 4) Matrix

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the electron transport chain may be found in the cytoplasmic membrane or the inner membrane of mitochondria

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What are the initial reactants which start the electron transport chain?

• During various steps in glycolysis and the citric acid cycle, the oxidation of certain intermediate precursor molecules causes the reduction of NAD+ to NADH + H+ and FAD to FADH2.

• NADH and FADH2 then transfer protons and electrons to the electron transport chain to produce additional ATPs from oxidative phosphorylation (is when phosphorylation is coupled with biological oxidation)

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Components of ETC

• NAD dehydrogenase• FMN, FAD• Ubiquinone or Co-enzyme Q (fat soluble not

protein)• Iron containing proteins (iron-sulfur Fe-S

protein) • Cytochromes (haemprotein) b, c, c1, aa3 • aa3 or cytochrome oxidase

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Components are present in the inner mitochondrial membrane as four complexes (cytochromes- electron carrier proteins)

• Complex-I NADH- Ubiquinone oxido-reductase

• Complex-II Succinate- Ubiquinone oxido-reductase

• Complex III Ubiquinol- Cytochrome c oxidoreductase

• Complex IV cytochromec (cyt) - Oxygen oxidoreductase

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electrons are transported to meet up with oxygen from respiration at the end of the chain. The overall electron chain transport reaction is: 2 H+ + 2 e+ + 1/2 O2 ---> H2O + energy

2 hydrogen ions, 2 electrons, and an oxygen molecule react to form as a product water with energy released in an exothermic reaction

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Reactions of Electron Transport Chain

• Electron carriers (NAD, FAD) carry the high energy electrons that produced in the first and second processes of cellular respiration (glycolysis &citric acid cycle) to a group of enzymes in inner membrane of mitochondria

• *NAD+ molecule accepts and transfers one hydride ion (H- i.e. one H+ & 2e-)

• *FMN or FAD or coenzyme Q accepts and donates 2H2 (2H+ & 2e) a time

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• *cytochrome or iron-sulfer protein molecule accepts and transfers only one electron but no H+

• Enzymes move electron along from one molecule to the other

• As the electrons (2e) passed, H+ ions are pumped to the outer membrane of mitochondria

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Formation of ATP in oxidative phosphorylation

• During the transfer of electrons energy is produced

• The energy is coupled to the formation of ATP by phosphorylation of ADP by the action of ATP synthase complex

• (ATP synthase complex converts this mechanical work into chemical energy by producing ATP

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• The transport of one pair of electrons from NADH to oxygen through the electron transport chain produces three molecules of ATP

• the transport of one pair of electrons from FADH2 to oxygen through the electron transport chain produces two molecules of ATP.

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• The ion gradient is used to run the ATP production by the electron transport phosphorylation (chemiosmosis)

• By the end electrons produced energy, electron carriers are back again the process continue

• Oxygen is the last electron acceptor • Water is the last product made O2 picks up

electron and combines with a H+ ions

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Reduction of oxygen to water

• Cytochrome oxidase (cyt aa3) the last cytochrome complex passes electron from cytochrome c to molecular oxygen

• O2 molecules must accept 4 electrons to reduce to water

• There are only two electrons per turn of ETC ETC must cycle twice to pass along 4 electrons to O2

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Each oxygen atom with two electrons accepts two protons thus a molecule of water resultthe reduction of oxygen to water result in production of about 300 ml of water/day (metabolic water)

molecule of water

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The end products of cellular respiration (glucose oxidation)

(glycolysis, citric acid cycle &ETC)• The over all equation of glucose oxidation=• C6H1206 +6O2→6CO2 +6H2O +ATP (36ATP-

2ATP)• Glycolysis = • 2ATP • 2NADH• 2Pyruvate

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• Products of pyruvate oxidation (to acetyl CA)• 2CO2• 2NADH• 2acetyl-CA• Products of Kerb’s cycle• 4CO2• 2FAD• 6NADH• 2ATP• 6 H+

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What are the final products of the chain

• Products of Electron transport chain• H2O• 3 ATP as free energy

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Gluconeogenesis (glucose synthesis)

• Production of glucose from non carbohydrates • The primary carbon skeletons used for

gluconeogenesis are derived from pyruvate, lactate, glycerol, and the amino acids alanine and glutamine.

• The liver is the major site of gluconeogenesis,

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Ref. Michael W King, PhD | © 1996–2012 themedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org

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Glycogen metabolism

• Glycogen is the major storage form of glucose in liver and muscle

• Metabolism involved• 1-glycogenesis• 2-glycogenolysis

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glycogenesis

• Is a pathway for formation of glycogen from glucose

• This process required energy in the form of ATP and UTP (uridine triphosphate)

• It occur in muscle and in liver when insulin/glucagonratio

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Reactions of glycogenesis

• 1-Phosphorylated of glucos to glucose 6-phosphate (hexokinase in muscles and glucokinase in liver

• 2-glucose 6-phosphate is converted to glucose 1-phosphate (phosphoglucomutase)

• 3-glucose 1-phosphate react with uridine triphosphate to form active nucleotide uridine diphosphate glucose (UDP-GLc) by the action of UDP-glucose pyrophosphorylase.

• Pyrophosphate (PiPi)is the second product of the reaction, is hydrolyzed to two inorganic phosphate by the action of pyrophosphatase

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• 4-pre-existing glycogen molecule must be present to start reaction 4

• By the action of enzyme glycogen synthase the C1 of the glucose of UDP-GLc forms a glycosidic bond with C4 of glucose residue of the re-existing glycogen (glycogen primer) liberating uridine diphosphate (UDP)

• 5-a new alfa-1-4 linkage is formed between carbon 1 of incoming glucose and carbon 4 of the terminal glucose of the glycogen primer

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• 6-when the chain lengthened to a minimum of 11 residues a second enzyme (branching enzyme) amylo-1,4 to 1,6-transglucosidase transfers a part of the 1,4-chain minimum length of glucose residues to a neighboring chain to form alpha 1,6-linkage (branching point of the molecule)

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Glycogenolysis

• Glycogenolysis is the process of degradation of glycogen to glucose 6-phosphate (muscle) and glucose (liver)

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Reactions of glycogenolysis

• 1-phosphorolysis of alpha 1,4-glycosidic bonds of glycogen to yield glucose 1-phosphate and residual glycogen molecule

• This reaction is catalyzed by glycogen phosphrylase• 2-by the action of phosphorylase, glucan

transferase and de-branching enzyme leads to complete breakdown of glycogen with the formation of glucose 1-phosphate and free glucose

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• 3-glucose 1-phosphate is converted to glucose 6-phosphate by phosphoglucomutase

• This is a reversible reaction• 4-in the liver specific enzyme glucose 6-

phosphatase removes phosphate from glucose 6-phosphate and free glucose which in turn diffuses from the cell to the blood

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Pentose phosphate pathway

• Known as hexose monophosphate shunt, phosphgluconate pathway

• It is the pathway for formation of pentose (5C) sugar from hexose sugar (6C)

• It is a multi-cyclic process in which three molecules of glucose 6-phosphate yeilds;

• -3 molecules of CO2

• -3 molecules of 5-carbon sugar

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The primary functions of Pentose phosphate pathway

• The primary functions of this pathway are: • 1. To generate reducing equivalents, in the form of

NADPH, for reductive biosynthesis reactions within cells.

• 2. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids.

• 3. metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates.

Ref. 1996–2012 themedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org

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Location of pentose phosphate pathway

• Main site of Pentose phosphate pathwayin cytosol due to the presence of the enzymes

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Reaction of pentose phosphate pathway

• Two phases for the reaction; • 1-oxidative irreversible phase

• 2-non-oxidative reversible phase

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Ref. Michael W King, PhD | © 1996–2012 themedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org

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reaction 1-oxidative irreversible phase of Pentose phosphate pathway

• 1-dehydrogenation of glucose 6-phosphate to 6-phospho-glucono-lactone

• Enzyme glucose 6-phosphate dehydrogenase (NADP dependent enzyme)

• 2- 6-phospho-glucono-lactone is hydrolyzed by 6-phospho-gluconolactone hydrolase to 6-phosphogluconat

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• 3- 6-phosphogluconat undergo oxidative decarboxylation by the action of 6-phosph-gluconate dehydrogenase (NADP is needed)

• The final product are of the oxidative irreversible phase;

• -ribulose 5-phosphate• -CO2

• -second molecule of NADPH

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reaction 2-non-oxidative reversible phase of Pentose phosphate pathway

• ribulose 5-phosphate is converted back to glucose 6-phosphate by sequence reactions

• stage 4 involved two enzymes; • 1-ribulose 5-phosphate 3-epimerase; alter

configuration of C3 forming epimer xylulose 5-phosphate (ketopentose)

• 2-ribose 5-phosphate ketoisomerase; convert ribulose 5-phosphate to aldopentose, ribose 5-phosphate

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Ref, Michael W King, PhD | © 1996–2012 hemedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org The primary enzymes involved in the non-oxidative steps of are transaldolase and transketolase:

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• 5-transketolase which transfers the two carbon 1,2 of keto to aldehyde carbon of aldose sugar

• This reaction converts an aldose to ketose TPP are required as co-enzyme additional to Mg2+

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• 6-transaldolase transfer three carbon dihydroxyacetone

• Transaldolase transfers 3 carbon groups and thus is also involved in a rearrangement of the carbon skeletons of the substrates of the PPP. The transaldolase reaction involves Schiff base formation between the substrate and a lysine residue in the enzyme.

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Final products of pentose phosphat shunt

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References

• National Center for Biotechnology Information, U.S. National Library of Medicine8600 Rockville Pike, BethesdaMD, 20894USA

• Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) Enzyme Nomenclature

• Michael W King, PhD | © 1996–2012 themedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org

• D. Voet, J. G. Voet, Biochemistry, second edition ed., John Wiley &• Sons, New York, 1995• National Center for Biotechnology Information, U.S. National Library of Medicine8600

Rockville Pike, BethesdaMD, 20894USA• Sareen Gropper, Jack Smith and James Groff, Advanced Nutrition and Human Metabolism,

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New York, NY: Oxford University Press. ISBN 978-0-19-960563-7.

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• Koppenol, W. H. (2002). "Naming of New Elements (IUPAC Recommendations 2002)" (PDF). Pure and Applied Chemistry 74 (5): 787–791. doi:10.1351/pac200274050787. http://media.iupac.org/publications/pac/2002/pdf/7405x0787.pdf.

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Appleton and Lange , twent fifth edition • Cooper GM 2000. The Central Role of Enzymes as Biological CatalystsThe Cell: A Molecular Approach.

2nd edition. Sunderland (MA): Sinauer Associates; 2000• Campbell, Neil A.; Brad Williamson; Robin J. Heyden (2006). Biology: Exploring Life. Boston,

Massachusetts: Pearson Prentice Hall• A. Burtis, Edward R. Ashwood, Norbert W. Tietz (2000), Tietz fundamentals of clinical chemistry • Maton, Anthea; Jean Hopkins, Charles William McLaughlin, Susan Johnson, Maryanna Quon Warner,

David LaHart, Jill D. Wright (1993). Human Biology and Health. Englewood Cliffs, New Jersey, USA: Prentice Hall. pp. 52–59

• Maitland, Jr Jones (1998). Organic Chemistry. W W Norton & Co Inc (Np). p. 139. ISBN 0-393-97378-6. • Nelson DL, Cox MM (2005). Lehninger's Principles of Biochemistry (4th ed.). New York, New York: W. H.

Freeman and Company.• Matthews, C. E.; K. E. Van Holde; K. G. Ahern (1999) Biochemistry. 3rd edition. Benjamin Cummings.• http://wiki.answers.com/Q/What_is_dehydration_synthesis#ixzz2BuiK645