CHAPTER 14 Glucose Utilization and

Biosynthesis

– Harnessing energy from glucose via glycolysis

– Fermentation under anaerobic conditions

– Synthesis of glucose from simpler compounds: gluconeogenesis

– Oxidation of glucose in pentose phosphate pathway

Problems:5, 6, 9, 10, 13, 14, 17, 20, 22, and 26http://ebooks.bfwpub.com/lehninger5e

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Central Importance of Glucose

• Glucose is an excellent fuel– Yields good amount of energy upon oxidation

– Can be e!ciently stored in the polymeric form

– Many organisms and tissues can meet their energy needs on glucose only

• Glucose is a versatile biochemical precursor– Bacteria can use glucose supply metabolic

intermediates used to build the carbon skeletons of:

• All the amino acids

• Membrane lipids

• Nucleotides in DNA and RNA

• Cofactors needed for the metabolism 2

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Four Major Pathways of Glucose Utilization

• When there’s plenty of excess energy, glucose can be

stored in the polymeric form (starch, glycogen)

• Short-term energy needs are met by oxidation of

glucose via glycolysis

• Pentose phosphate pathway generates NADPH that is

used for detoxification, and for the reductive

biosynthesis of lipids and nucleotides and generates

Pentose phosphate

• Structural polysaccharides (e.g. in cell walls of bacteria,

fungi, and plants) are derived from glucose

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Glycolysis: Importance

• Glycolysis is a sequence of enzyme-catalyzed reaction

by which glucose is converted into pyruvate

• Pyruvate can be further aerobically oxidized (Citric Acid cycle

to yield carbon dioxide and water)

• Pyruvate can be used as a precursor in biosynthesis

• In the process, some of the oxidation free energy in

captured by the synthesis of ATP and NADH

• Research of glycolysis played a large role in the

development of modern biochemistry

– Understanding the role of coenzymes

– Discovery of the pivotal role of ATP

– Development of methods for enzyme purification

– Inspiration for the next generations of biochemists 5

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Glycolysis: Overview

• In the evolution of life, glycolysis probably was one of the earliest energy-yielding pathways

• It developed before photosynthesis, when the atmosphere was still anaerobic

• Thus, the task upon early organisms was how to extract free energy from glucose anaerobically?

•The solution

–Activate it first by transferring couple of phosphates to it

–Collect energy later from the high-energy metabolites of the activated glucose 6

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Glycolysis: The Preparatory Phase

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For each molecule of glucose that passes through the preparatory phase (a), two molecules of glyceraldehyde 3-phosphate are formed

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Glycolysis: The Payo" Phase

Pyruvate is the end product of the second phase of glycolysis. For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP per molecule of glucose converted to pyruvate. The numbered reaction steps are catalyzed by the enzymes listed on the right. Keep in mind that each phosphoryl group, represented here as P, has two negative charges (—PO3

2–).

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Triose phosphate isomerase

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1.The Hexokinase Reaction

• The first step, phosphorylation of glucose, is

catalyzed by hexokinase in eukaryotes, and by

glucokinase in prokaryotes

• Nucleophilic oxygen at C6 of glucose attacks

the last (!) phosphorous of ATP

• Bound Mg++ facilitates this process by

stabilizing the negative charge in the

transition state

• This process uses the energy of ATP

• This process is irreversible13

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1.

Irreversible

Kinases catalyze phosphorylation of molecules by ATP.

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2. Phosphohexose Isomerization

• An aldose can isomerize into ketose via an

enediol intermediate

• The isomerization is catalyzed by the active-

site glutamate

• In one step, ionized Glu acts as a general

base to abstract the proton from C2 and

generate the enediol

• In the next step, protonated Glu acts as a

general acid to re-protonate enediol at C1

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2.

Isomerases catalyze the transformation of compounds into their positional isomers.

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2. Mechanism of Phosphohexose Isomerase

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2.

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2.

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2.

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3. The Second Priming Reaction; The First Commitment

• ATP is the donor of the second phosphate group

• This is an irreversible step

• The product, fructose 1,6-bisphosphate is committed to become pyruvate and yield energy

• Phosphofructokinase-1 is negatively regulated by ATP

– Do not burn glucose if there is plenty of ATP

– This process is irreversible

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3.

Irreversible

!G’0 = -14.2 kJ/mol

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4. Aldolases Cleave 6-Carbon Sugars

• The reverse process is the familiar aldol condensation

• Animal and plant aldolases employ covalent catalysis

• Fungal and bacterial aldolases employ metal ion catalysis

• Aldolases catalyze reversible aldol condensation

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DAP GAP

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"G’0 is positive but

the reactants are present in low concentrations.

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5. Triose Phosphate Interconversion

• Aldolase creates two triose phosphates: DAP

and GAP

• Only GAP is the substrate for the next

enzyme (Step 6)

• DAP is converted enzymatically to GAP

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5.

GAP is continuously used up.27

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6. Glyceraldehyde 3-Phosphate Dehydrogenase

Reaction • First energy-yielding step in glycolysis

• Oxidation of aldehyde with NAD+ gives NADH

• Phosphorylation yields an high-energy reaction product

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6.

Very high energy of hydrolysis:-49.3 kJ/mol

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7. First Substrate-Level Phosphorylation

• 1,3-bisphosphoglycerate is a high-energy

compound that can donate the phosphate

group to ADP to make ATP

• The reaction is reversible, the reverse process

transfer of phosphate from ATP to

phosphoglycerate

• Kinases are enzymes that transfer phosphate

groups from molecules like ATP to various

substrates

• Substrate level phosphorylation31

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7.

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8. Conversion of 3-Phosphoglycerate to 2-

Phosphoglycerate• This is a reversible isomerization reaction

• Enzymes that shift functional groups around are called mutases

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8.

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8. Mechanism of the Phosphoglycerate Mutase

Reaction• Phosphoglycerate mutase employs covalent

catalysis

• One of the active site histidines is post-

translationally modified to phosphohistidine

• Phosphohistidine donates its phosphate to O2

before retrieving another phosphate from O3

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8. Mechanism of the Phosphoglycerate Mutase

Reaction• Notice that the phosphate from the substrate

ends up bound to the enzyme at the end of the reaction

• The two negative charges in the product are fairly close now but 2-phosphoglycerate is not good enough phosphate donor

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9. Dehydration of 2-Phosphoglycerate

• The goal here is to create a better phosphoryl donor

• Loss of phosphate from 2-phosphoglycerate would merely give a secondary alcohol with no further stabilization.

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9.

Energy to transfer phosphate from 1. = -17.6 kJ/molEnergy to transfer phosphate from 2= -61.9 kJ/mol

1 2

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10 Second Substrate-Level Phosphorylation

• … but loss of phosphate from

phosphoenolpyruvate yields an enol that

tautomerizes into ketone

• The tautomerization e"ectively lowers the

concentration of the reaction product and

drives the reaction toward ATP formation

• Substrate level phosphorylation

• Irreversible reaction

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Irreversible

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Pyruvate Kinase is Subject to Regulation

• Pyruvate kinase requires divalent metals (Mg++ or Mn++) for activity

• Under physiological conditions, the activity of pyruvate kinase is limited by the level of Mg++

• When there is plenty of ATP, the Mg ions are sequestered by ATP; this slows down pyruvate kinase

• Increased concentration of metabolites in the glycolytic pathway slows down glucose utilization 44

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Glycolysis Occurs at Elevated Rates in Tumor

Cells

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The anaerobic metabolism of glucose in tumor cells yields far less ATP (2 per glucose) than the complete oxidation to CO2 that

takes place in healthy cells under aerobic conditions (~30 ATP per glucose), so a tumor cell must consume much more glucose to produce the same amount of ATP. Glucose transporters and most of the glycolytic enzymes are overproduced in tumors. Compounds that inhibit hexokinase, glucose 6-phosphate dehydrogenase, or transketolase block ATP production by glycolysis, thus depriving the cancer cell of energy and killing it.

glucosetransporters

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Glycolysis:Glycolysis is a near-universal pathway by which a glucose molecule is oxidized to two molecules of pyruvate, with energy conserved as ATP and NADH.

All 10 glycolytic enzymes are in the cytosol, and all 10 intermediates are phosphorylated compounds of three or six carbons.

In the preparatory phase of glycolysis, ATP is invested to convert glucose to fructose 1,6-bisphosphate. The bond between C-3 and C-4 is then broken to yield two molecules of triose phosphate.

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In the payoff phase, each of the two molecules of glyceraldehyde 3-phosphate derived from glucose undergoes oxidation at C-1; the energy of this oxidation reaction is conserved in the form of one NADH and two ATP per triose phosphate oxidized. The net equation for the overall process is

Glucose + 2NAD+ + 2ADP + 2Pi " 2 pyruvate +

2NADH + 2H+ 2ATP + 2H2O

Glycolysis is tightly regulated in coordination with other energy-yielding pathways to assure a steady supply of ATP.

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Under Anaerobic Conditions, Animals Reduce Pyruvate to

Lactate

• During strenuous exercise, lactate builds up in the muscle

• The acidification of muscle prevents its continuous strenuous work

• The lactate can be transported to liver and converted to glucose there

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Under Anaerobic Conditions, Yeast Ferments Glucose to

Ethanol

• Both steps require cofactors

– Mg++ and thiamine pyrophosphate in pyruvate decarboxylase

– Zn++ and NAD+ in alcohol dehydrogenase

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Mechanism of Aldehyde Reduction by Alcohol

Dehydrogenase

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Feeder Pathways for Glycolysis

Other carbohydrates undergo glycolysis when they are transformed into a glycolytic intermediate.

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Endogenous Glycogen and Starch Are Degraded by Phosphorolysis

Glycogen stored in animal tissues (primarily liver and skeletal muscle), in microorganisms, or in plant tissues can be mobilized for use within the same cell by a phosphorolytic reaction catalyzed by glycogen phosphorylase (starch phosphorylase in plants).

These enzymes catalyze an attack by Pi on the (#1"4)

glycosidic linkage that joins the last two glucose residues at a nonreducing end, generating glucose 1-phosphate and a polymer one glucose unit shorter. Phosphorolysis preserves some of the energy of the glycosidic bond in the phosphate ester glucose 1-phosphate.

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Glucose 1-phosphate produced by glycogen phosphorylase is converted to glucose 6-phosphate by phosphoglucomutase, which catalyzes the reversible reaction:Glucose 1-phosphate glucose 6-phosphate 60

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In glycolysis it is an irreversible reaction:

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WORKED EXAMPLE 14-1 Energy Savings for Glycogen Breakdown by Phosphorolysis. Calculate the energy savings (in ATP molecules per glucose monomer) achieved by breaking down glycogen by phosphorolysis rather than hydrolysis to begin the process of glycolysis.

Solution: Phosphorolysis produces a phosphorylated glucose (glucose 1-phosphate), which is then converted to glucose 6-phosphate—without expenditure of the cellular energy (1 ATP) needed for formation of glucose 6-phosphate from free glucose. Thus only 1 ATP is consumed per glucose monomer in the preparatory phase, compared with 2 ATP when glycolysis starts with free glucose. The cell therefore gains 3 ATP per glucose monomer (4 ATP produced in the payoff phase minus 1 ATP used in the preparatory phase), rather than 2—a saving of 1 ATP per glucose monomer.

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Feeder Pathways for Glycolysis

•Endogenous glycogen and starch, storage forms of glucose, enter glycolysis in a two-step process. Phosphorolytic cleavage of a glucose residue from an end of the polymer, forming glucose 1-phosphate, is catalyzed by glycogen phosphorylase or starch phosphorylase. Phosphoglucomutase then converts the glucose 1-phosphate to glucose 6-phosphate, which can enter glycolysis.

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•Ingested polysaccharides and disaccharides are converted to monosaccharides by intestinal hydrolytic enzymes, and the monosaccharides then enter intestinal cells and are transported to the liver or other tissues.

•A variety of D-hexoses, including fructose, galactose, and mannose, can be funneled into glycolysis. Each is phosphorylated and converted to glucose 6-phosphate, fructose 6-phosphate, or fructose 1-phosphate.Conversion of galactose 1-phosphate to glucose 1-phosphate also occurs.

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Gluconeogenesis: Precursors for Carbohydrates

• Tissues that depend solely on glucose for metabolic energy:

brain, nervous system, erythrocytes, testes, renal medulla and embryonic tissue.

• Sometimes there is not enough glucose stored as glycogen- then glucose is synthesized from non carbohydrate precursors.

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What Is Gluconeogenesis, and How Does It Operate?

Synthesis of "new glucose" from common metabolites

• Humans consume 160 g of glucose per day

• 75% of that is in the brain

• Body fluids contain only 20 g of glucose

• Glycogen stores yield 180-200 g of glucose

• So the body must be able to make its own glucose

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The pathway from phosphoenolpyruvate to glucose 6-phosphate is common to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants. The path from pyruvate to phosphoenolpyruvate leads through oxaloacetate, an intermediate of the citric acid cycle. Any compound that can be converted to either pyruvate or oxaloacetate can therefore serve as starting material for gluconeogenesis. This includes alanine and aspartate, which are convertible to pyruvate and oxaloacetate, respectively, and other amino acids that can also yield three- or four-carbon fragments, the so-called glucogenic amino acids (Table 14-4). Plants and photosynthetic bacteria are uniquely able to convert CO2 to carbohydrates

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We will focus on gluconeogenesis in mammalian liver

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Glucogenic-converted into glucose

Ketogenic-converted into ketones

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Substrates for Gluconeogenesisin mammals

Pyruvate, lactate, glycerol, amino acids and all TCA (tricarboxylic acid cycle)

intermediates can be utilized

• Fatty acids cannot!

• Why?

• Most fatty acids yield only acetyl-CoA

• Acetyl-CoA (through TCA cycle) cannot provide for net synthesis of sugars

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The Central Relationship of the Citric Acid Cycle to

Catabolism

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CoA CO2

+ +NAD+ NADH

Pyruvate acetyl-CoA

Pyruvate from glycolysis is converted to acetyl-CoA Via Pyruvate dehydrogenase. This is an Irreversible reaction. The acetyl-CoA then goe into the citric acid cycle.

Animals cannot get to pyruvate from acetyl-CoA

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Opposing pathways of glycolysis and gluconeogenesis in rat liver.

The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here.

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Gluconeogenesis

• Occurs mainly in liver and kidneys

• Not the mere reversal of glycolysis for 2 reasons:

– Energetics must change to make gluconeogenesis favorable (delta G of glycolysis = -74 kJ/mol, or about that)

– Reciprocal regulation: Gluconeogenesis must turn one on and the other o".

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Gluconeogenesis

• Seven steps of glycolysis are retained:– Steps 2 and 4-9

• Three steps are replaced:– Steps 1, 3, and 10 (the regulated

steps!)• The new reactions provide for a

spontaneous pathway ("G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation

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1replaced

3replaced

2

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5

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10replaced

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9

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Steps 1, 3, and 10 are replaced. These are the non-reversible, regulated steps. In cells these reactions have large, negative free energy changes. The other 7 reaction steps have a "G

near zero.

Steps 1, 3 and 10 are replaced by a new set of reactions catalyzed by enzymes. These reactions are exergonic and irreversible in the direction of glucose synthesis.

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Synthesis of Oxaloacetate

• Conversion of pyruvate to energy-rich

phosphoenolpyruvate requires two energy-

consuming steps

• In the first step, pyruvate is transported into

mitochondria and converted into oxaloacetate

by pyruvate carboxylase

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10 -10B

-10A

"G’0 = -31.4 kJ/mol + 0.9 kJ/mol"G = -16.7 kJ/mol - 25 kJ/mol

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Pyruvate CarboxylaseMitochondrial Enzyme

Pyruvate is converted to oxaloacetate -10A

• The reaction requires ATP and bicarbonate as substrates

• Coenzyme Biotin is covalently linked to an active site lysine

• Acetyl-CoA is an allosteric activator

• Regulation: when ATP or acetyl-CoA are high, pyruvate enters gluconeogenesis

• Acetyl-CoA is produced by oxidation of fatty acids and signals that fatty acids are available as fuel.

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Acetyl-CoA and ATP signal that energy is abundant.

Metabolites are converted into glucose and maybe even into glycogen.

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In mitochondria, pyruvate is converted to oxaloacetate in a biotin-requiring reaction catalyzed by pyruvate carboxylase.

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Pyruvate carboxylase is a compartmentalized reaction. Pyruvate is converted to oxaloacetate in the mitochondria. Because oxaloacetate cannot be transported across the mitochondrial membrane, it must be reduced to malate, transported to the cytosol, and then oxidized back to oxaloacetate ( -10B) before gluconeogenesis can continue.

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Oxaloacetate Picks Up Phosphate from GTP

• The phosphoenolpyruvate carboxykinase

reaction occurs either in the cytosol or the

mitochondria.

- 10B

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In the cytosol, oxaloacetate is converted to phosphoenolpyruvate by PEP carboxykinase. The CO2

incorporated in the pyruvate carboxylase reaction is lost here as CO2. The decarboxylation leads to a rearrangement of electrons

that facilitates attack of the carbonyl oxygen of the pyruvate moiety on the phosphate of GTP.

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Pyruvate + HCO3- + H+ # oxaloacetate + ADP + Pi

Oxaloacetate + NADH + H+ $ l-malate + NAD+

l-malate + NAD+ # oxaloacetate + NADH + H+

Oxaloactetate + GTP $ PEP + CO2 +GDP

Pyruvate + ATP + GTP + HCO3- # PEP + ADP + GDP + Pi

+CO2

"G’0 = + 0.9 kJ/mol"G = - 25 kJ/mol

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Fructose-1,6-bisphosphatase

Hydrolysis of F-1,6-bisPase to F-6-P

• Thermodynamically favorable - "G in

liver is -8.6 kJ/mol ("G’0 = -16.3 kJ/

mol)

• Allosteric regulation:

– citrate stimulates

– fructose-2,6-bisphosphate inhibits

– AMP inhibits

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A high level of AMP indicates that the energy is low and signals the need for ATP generation. Glycolysis is needed.

Conversely, high levels of ATP and citrate indicate that the energy is high and that biosynthetic intermediates are abundant. Glycolysis is nearly switched off and gluconeogenesis is promoted.

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3 -3

"G’0 = -16.3 kJ/mol

"G = -8.6 kJ/mol in

liver

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Glucose-6-PhosphataseConversion of Glucose-6-P to Glucose

• Presence of G-6-Pase in ER of liver and kidney cells makes gluconeogenesis possible

• Muscle and brain do not undergo gluconeogenesis

• G-6-P is hydrolyzed as it passes into the ER

• ER vesicles filled with glucose di"use to the plasma membrane, fuse with it and open, releasing glucose into the bloodstream.

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1 -1

" G’0 = -13.8 kJ/mol

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Glucose-6-phosphatase is localized in the endoplasmic reticulum membrane. Conversion of glucose-6-phosphate to glucose occurs during transport into the ER.

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The glucose-6-phosphatase reaction involves formation of a phosphohistidine intermediate.

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Glucose-6-phosphatase activity in controlled by the level of glucose-6-phosphate. This is substrate level control.

Activity is not under allosteric control.

When levels of glucose-6-phosphate are high, glycolysis switched off.

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Glycolysis:Glycolysis is a near-universal pathway by which a glucose molecule is oxidized to two molecules of pyruvate, with energy conserved as ATP and NADH.

All 10 glycolytic enzymes are in the cytosol, and all 10 intermediates are phosphorylated compounds of three or six carbons.

In the preparatory phase of glycolysis, ATP is invested to convert glucose to fructose 1,6-bisphosphate. The bond between C-3 and C-4 is then broken to yield two molecules of triose phosphate.

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In the payoff phase, each of the two molecules of glyceraldehyde 3-phosphate derived from glucose undergoes oxidation at C-1; the energy of this oxidation reaction is conserved in the form of one NADH and two ATP per triose phosphate oxidized. The net equation for the overall process is

Glucose + 2NAD+ + 2ADP + 2Pi " 2 pyruvate +

2NADH + 2H+ 2ATP + 2H2O

Glycolysis is tightly regulated in coordination with other energy-yielding pathways to assure a steady supply of ATP.

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Feeder Pathways for Glycolysis

•Endogenous glycogen and starch, storage forms of glucose, enter glycolysis in a two-step process. Phosphorolytic cleavage of a glucose residue from an end of the polymer, forming glucose 1-phosphate, is catalyzed by glycogen phosphorylase or starch phosphorylase. Phosphoglucomutase then converts the glucose 1-phosphate to glucose 6-phosphate, which can enter glycolysis.

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•Ingested polysaccharides and disaccharides are converted to monosaccharides by intestinal hydrolytic enzymes, and the monosaccharides then enter intestinal cells and are transported to the liver or other tissues.

•A variety of D-hexoses, including fructose, galactose, and mannose, can be funneled into glycolysis. Each is phosphorylated and converted to glucose 6-phosphate, fructose 6-phosphate, or fructose 1-phosphate.Conversion of galactose 1-phosphate to glucose 1-phosphate also occurs.

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Gluconeogenesis

• Gluconeogenesis is a ubiquitous multistep process in which glucose is produced from lactate, pyruvate, or oxaloacetate, or any compound (including citric acid cycle intermediates) that can be converted to one of these intermediates.

Seven of the steps in gluconeogenesis are catalyzed by the same enzymes used in glycolysis; these are the reversible reactions.

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•Three irreversible steps in glycolysis are bypassed by reactions catalyzed by gluconeogenic enzymes: (1) conversion of pyruvate to PEP via oxaloacetate, catalyzed by pyruvate carboxylase and PEP carboxykinase;

(2) dephosphorylation of fructose 1,6-bisphosphate by FBPase-1; and

(3) dephosphorylation of glucose 6-phosphate by glucose 6-phosphatase.

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•Formation of one molecule of glucose from pyruvate requires 4 ATP, 2 GTP, and 2 NADH; it is expensive.

•In mammals, gluconeogenesis in the liver, kidney, and small intestine provides glucose for use by the brain, muscles, and erythrocytes.

•Pyruvate carboxylase is stimulated by acetyl-CoA, increasing the rate of gluconeogenesis when the cell has adequate supplies of other substrates (fatty acids) for energy production.

•Animals cannot convert acetyl-CoA derived from fatty acids into glucose; plants and microorganisms can.

•Glycolysis and gluconeogenesis are reciprocally regulated to prevent wasteful operation of both pathways at the same time.

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Pentose Phosphate Pathway

• The main goals are to produce NADPH for anabolic reactions and ribose 5-phosphate for nucleotides

oxidations

NAD+ + 2e- + 2H+ # NADH + H+

NADP+ + 2e- + 2H+ # NADPH + H+

reductions 105

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Pentose Phosphate Pathway

• The main goals are to produce NADPH for anabolic reactions and ribose 5-phosphate for nucleotides

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Can Glucose Provide Electrons for Biosynthesis?

Pentose Phosphate Pathwayaka hexose monophosphate shunt

• Provides NADPH for biosynthesis • Produces ribose-5-P • Two oxidative processes followed by

five non-oxidative steps • Operates mostly in cytoplasm of liver

and adipose cells • NADPH is used in cytosol for fatty acid

synthesis

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General scheme of the pentose phosphate pathway.

NADPH formed in the oxidative phase is used to reduce glutathione, GSSG and to support reductive biosynthesis. The other product of the oxidative phase is ribose 5-phosphate, which serves as a precursor for nucleotides, coenzymes, and nucleic acids.

In cells that are not using ribose 5-phosphate for biosynthesis, the nonoxidative phase recycles six molecules of the pentose into five molecules of the hexose glucose 6-phosphate, allowing continued production of NADPH and converting glucose 6-phosphate (in six cycles) to CO2.

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Oxidative phase:The end products are ribose 5-phosphate, CO2, and NADPH.

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Glucose-6-phosphate is oxidized:The carbon oxidation number increased from +1to +3.

Equilibrium isIn favor of NADPHformation

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The lactone is hydrolyzed to the free acid 6-phosphogluconate by a specific lactonase.

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6-phosphogluconate undergoes oxidation and decarboxylation by 6-phosphogluconate dehydrogenase to form the ketopentose ribulose 5-phosphate; the reaction generates a second molecule of NADPH.

+2

+4

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Phosphopentose isomerase converts ribulose 5-phosphate to its aldose isomer, ribose 5-phosphate. In some tissues, the pentose phosphate pathway ends at this point, and its overall equation is:

The net result is the production of NADPH, a reductant for biosynthetic reactions, and ribose 5-phosphate, a precursor for nucleotide synthesis.

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The Nonoxidative Phase Recycles Pentose Phosphates to Glucose 6-Phosphate:

In tissues that require primarily NADPH, the pentose phosphates produced in the oxidative phase of the pathway are recycled into glucose 6-phosphate. In this nonoxidative phase, ribulose 5-phosphate is first epimerized to xylulose 5-phosphate.

Epimers differ in configuration at one asymmetric center in compounds that have two or more asymmetric centers.

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In a series of rearrangements of the carbon skeletons 6 five-carbon sugar phosphates are converted to 5 six-carbon sugar phosphates, completing the cycle and allowing continued oxidation of glucose 6-phosphate with production of NADPH. Continued recycling leads ultimately to the conversion of glucose 6-phosphate to six CO2.

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Two enzymes unique to the pentose phosphate pathway act in these interconversions of sugars: transketolase and transaldolase. Transketolase catalyzes the transfer of a two-carbon fragment from a ketose donor to an aldose acceptor. Next, transaldolase removes a 3 carbon fragment and condenses it with glyceraldehyde-3-phosphate forminfg fructose-6-phosphate.

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NADPH Regulates Partitioning into Glycolysis vs. Pentose

Phosphate Pathway

• NADPH inhibits

glucose-6-phosphate

dehydrogenase

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Role of NADPH in regulating the partitioning of glucose 6-phosphate between glycolysis and the pentose phosphate pathway. When NADPH is forming faster than it is being used for biosynthesis and glutathione reduction (see Figure 14-20), [NADPH] rises and inhibits the first enzyme in the pentose phosphate pathway. As a result, more glucose 6-phosphate is available for glycolysis.

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Pentose Phosphate Pathway of Glucose Oxidation

•The oxidative pentose phosphate pathway (phosphogluconate pathway, or hexose monophosphate pathway) brings about oxidation and decarboxylation at C-1 of glucose 6-phosphate, reducing NADP+ to NADPH and producing pentose phosphates.

•NADPH provides reducing power for biosynthetic reactions, and ribose 5-phosphate is a precursor for nucleotide and nucleic acid synthesis. Rapidly growing tissues and tissues carrying out active biosynthesis of fatty acids, cholesterol, or steroid hormones send more glucose 6-phosphate through the pentose phosphate pathway than do tissues with less demand for pentose phosphates and reducing power.

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•Entry of glucose 6-phosphate either into glycolysis or into the pentose phosphate pathway is largely determined by the relative concentrations of NADP+ and NADPH.

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Chapter 14: Summary

• Glycolysis, a process by which cells can extract a

limited amount of energy from glucose under

anaerobic conditions

• Gluconeogenesis, a process by which cells can use a

variety of metabolites for the synthesis of glucose

• Pentose phosphate pathway, a process by which cells

can generate reducing power (NADPH) that is needed

for the biosynthesis of various compounds

In this chapter, we learned about:

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5. Energetics of the Aldolase Reaction

Aldolase catalyzes the glycolytic reaction:

Fructose 1,6-bisphosphate " glyceraldehyde 3-phosphate + dihydroxyacetone phosphate

The standard free-energy change for this reaction in the direction written is +23.8 kJ/mol. The concentrations of the three intermediates in the hepatocyte of a mammal are: fructose 1,6-bisphosphate,1.4 X10-5 M; glyceraldehyde 3-phosphate, 3 X10-6 M; and dihydroxyacetone phosphate, 1.6 X 10-5 M. At body temperature (370 C), what is the actual free-energy change for the reaction?

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DAP GAP

4

"G’0 is positive but

the reactants are present in low concentrations.

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6. Pathway of Atoms in Fermentation A “pulse-chase“ experiment using 14C-labeled carbon sources is carried out on a yeast extract maintained under strictly anaerobic conditions to produce ethanol.The experiment consists of incubating a small amount of 14C-labeled substrate (the pulse) with the yeast extract just long enough for each intermediate in the fermentation pathway to become labeled. The label is then “chased” through the pathway by the addition of excess unlabeled glucose. The chase effectively prevents any further entry of labeled glucose into the pathway.

(a)If [1-14C glucose (glucose labeled at C-1 with 14C) is used as a substrate, what is the location of 14C in the product ethanol? Explain.

Figure 14–6 illustrates the fate of the carbon atoms of glucose. C-1 (or C-6) becomes C-3 of glyceraldehyde 3-phosphate and subsequently pyruvate. When pyruvate is decarboxylated and reduced to ethanol, C-3 of pyruvate becomes the C-2 of ethanol (14CH3—CH2—OH).

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Triose phosphate isomerase

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(b) Where would 14C have to be located in the starting glucose to ensure that all the 14C activity is liberated as 14CO2 during fermentation to ethanol? Explain.

If all the labeled carbon from glucose is converted to 14CO2 during ethanol fermentation,

the original label must have been on C-3 and/or C-4 of glucose, because these are converted to the carboxyl group of pyruvate.

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9. Equivalence of Triose Phosphates 14C -Labeled glyceraldehyde 3-phosphate was added to a yeast

extract. After a short time, fructose 1,6-bisphosphate labeled with 14C at C-3 and C-4 was isolated.

What was the location of the 14C label in the starting

glyceraldehyde 3-phosphate? Where did the second 14C label in

fructose 1,6-bisphosphate come from? Explain.

Problem 1 outlines the steps in glycolysis involving fructose 1,6-

bisphosphate, glyceraldehyde 3-phosphate, and

dihydroxyacetone phosphate. Keep in mind that the aldolase

reaction is readily reversible and the triose phosphate isomerase

reaction catalyzes extremely rapid interconversion of its

substrates. Thus, the label at C-1 of glyceraldehyde 3-phosphate

would equilibrate with C-1 of dihydroxyacetone phosphate (#G’0

=+7.5 kJ/mol). Because the aldolase reaction has #G’0 =- 23.8

kJ/mol in the direction of hexose formation, fructose 1,6-

bisphosphate would be readily formed, and labeled in C-3 and

C-4 (see Fig. 14–6).133

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10. Glycolysis Shortcut Suppose you discovered a mutant yeast whose glycolytic pathway was shorter because of the presence of a new enzyme catalyzing the reaction:

Would shortening the glycolytic pathway in this way benefit the cell? Explain.Answer Under anaerobic conditions, the phosphoglycerate kinase and pyruvate kinase reactions are essential. The shortcut in the mutant yeast would bypass the formation of an acyl phosphate by glyceraldehyde 3-phosphate dehydrogenase and therefore would not allow the formation of 1,3-bisphosphoglycerate. Without the formation of a substrate for 3-phosphoglycerate kinase, no ATP would be formed. Under anaerobic conditions, the net reaction for glycolysisnormally produces 2 ATP per glucose. In the mutant yeast, net production of ATPwould be zero and growth could not occur. Under aerobic conditions, however, because the majority of ATP formation occurs via oxidative phosphorylation, the mutation would have no observable effect.

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13. Free-Energy Change for Triose Phosphate Oxidation

The oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate dehydrogenase, proceeds with an unfavorable equilibrium constant

(K’eq 0.08; !G’0 =+ 6.3 kJ/mol), yet the flow through this point in the

glycolytic pathway proceeds smoothly. How does the cell overcome the unfavorableequilibrium?

Answer In organisms, where directional flow in a pathway is required, exergonic reactions are coupled to endergonic reactions to overcome unfavorable free-energy changes. The endergonic glyceraldehyde 3-phosphate dehydrogenase reaction is followed by the phosphoglycerate kinase reaction, which rapidly removes the product of the former reaction. Consequently, the dehydrogenase reaction does not reach equilibrium and its unfavorable free-energy change is

thus circumvented. The net !G’0 of the two reactions, when coupled, is -18.5 kJ/mol=+ 6.3 kJ/mol =- 12.2 kJ/mol.

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6.

Very high energy of hydrolysis:-49.3 kJ/mol

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14. Arsenate Poisoning Arsenate is structurally and chemically similar to inorganic phosphate (Pi), and many enzymes that require phosphate will also use arsenate. Organic compounds of arsenate are less stable than analogous phosphate compounds, however. For example, acyl arsenates decompose rapidly by hydrolysis:

On the other hand, acyl phosphates, such as 1,3-bisphosphoglycerate, are more stable and undergo further enzyme-catalyzed transformation in cells.(a)Predict the effect on the net reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase if phosphate were replaced by arsenate.In the presence of arsenate, the product of the glyceraldehyde 3-phosphate dehydrogenase reaction is 1-arseno-3-phosphoglycerate, which nonenzymatically decomposes to 3- phosphoglycerate and arsenate; the substrate for the phosphoglycerate kinase is therefore bypassed

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(b) What would be the consequence to an organism if arsenate were substituted for phosphate? Arsenate is very toxic to most organisms. Explain why.

No ATP can be formed in the presence of arsenate because 1,3-bisphosphoglycerate is not formed. Under anaerobic conditions, this would result in no net glycolytic synthesis of ATP. Arsenate poisoning can be used as a test for the presence of an acyl phosphate intermediate in a reaction pathway.

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17. Synthesis of Glycerol Phosphate The glycerol 3-phosphate required for the synthesis of glycerophospholipids can be synthesized from a glycolytic intermediate. Propose a reaction sequence for this conversion.Answer Glycerol 3-phosphate and dihydroxyacetone-3-phosphate differ only at C-2. A dehydrogenase with the cofactor NADH acting on dihydroxyacetone-3-phosphate would form glycerol-3-phospate.

In fact, the enzyme glycerol 3-phosphate dehydrogenase catalyzes this reaction (seeFig. 21–17).

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20. Pathway of Atoms in Gluconeogenesis A liver extract capable of carrying out all the normal metabolic reactions of the liver is briefly incubated in separate experiments with the following 14C-labeled precursors:

Trace the pathway of each precursor through gluconeogenesis. Indicate the location of 14C in all intermediates and in the product, glucose.Answer(a) In the pyruvate carboxylase reaction, 14CO2 is added to pyruvate

to form [4-14C]oxaloacetate, but the phosphoenolpyruvate carboxykinase reaction removes the same CO2 in the next step.

Thus, 14C is not (initially) incorporated into glucose.145

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In mitochondria, pyruvate is converted to oxaloacetate in a biotin-requiring reaction catalyzed by pyruvate carboxylase.

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22. Relationship between Gluconeogenesis and GlycolysisWhy is it important that gluconeogenesis is not the exact reversal of glycolysis?

Answer If gluconeogenesis were simply the reactions of glycolysis in reverse, the process would be energetically unfeasible (highly endergonic), because of the three reactions with large, negative standard free-energy changes in the catabolic (glycolytic) direction.

Furthermore, if the same enzymes were used for all reactions in the two pathways, it would be impossible to regulate the two processes separately; anything that stimulated (or inhibited) the forward reaction for a given enzyme would stimulate (or inhibit) the reverse reaction to the same extent. 149

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26. Blood Lactate Levels during Vigorous Exercise The concentrations of lactate in blood plasma before, during, and after a 400 m sprint are shown in the graph.

(a)What causes the rapid rise in lactate concentration?Rapid depletion of ATP during strenuous muscular exertion causes the rate of glycolysis to increase dramatically, producing higher cytosolic concentrations of pyruvate and NADH; lactate dehydrogenase converts these to lactate and NAD+ (lactic acid fermentation).

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(b) What causes the decline in lactate concentration after completion of the sprint? Why does the decline occur more slowly than the increase?When energy demands are reduced, the oxidative capacity of the mitochondria is again adequate, and lactate is transformed to pyruvate by lactate dehydrogenase, and the pyruvate is converted to glucose. The rate of the dehydrogenase reaction is slower in this direction because of the limited availability of NAD and because the equilibrium of the reaction is strongly in favor of lactate (conversion of lactate to pyruvate is energy requiring).

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(c) Why is the concentration of lactate not zero during the resting state?

The equilibrium of the lactate dehydrogenase reactionis strongly in favor of lactate. Thus, even at very low concentrations of NADH and pyruvate, there is a significant concentration of lactate.

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