陳泰源 博士

中央研究院生物化學研究所

2007/5/1 National Taiwan Normal University

Storage

Pentose phosphate pathway (oxidation)

Glycolysis (oxidation)

Glycogen, Starch, Sucrose

PyruvateRibose 5-phosphate

14.1 Glycolysishttp://www.tcd.ie/Biochemistry/IUBMB-Nicholson/swf/glycolysis.swf

Glycolysis• The glycolytic breakdown of glucose is the

sole source of metabolic energy in some mammalian tissues and cell types (erythrocytes, renal medulla( 腎髓 ), brain, and sperm.

• Fermentation is a general term for the anaerobic degradation of glucose or other organic nutrients to obtain energy (ATP).

• The pathway are conserved evolutionary in vertebrates, yeast and spinach.

1. phosphorylation of glucose

G 6-P

• Kinase: (transferases): catalyze the transfer of the terminal phosphoryl group from ATP to some acceptor nucleophile.

• Hepatocytes (Liver cell) also contain a form of hexokinase called hexokinase IV or glucokinase (more specific for glucose and differs from other hexokinase in kinetic and regulatory properties

• Isozymes: Two enzymes that catalyze the same reaction but are encoded in different genes

2. Isomerization of glucose 6-phosphate

G 6-P F 6-P

The phosphohexose isomerase reaction

• the ring opening and closing reactions (steps 1 and 4 ) are catalyzed by an active-site His residue.

• The movement of the proton between C-2 and C-1 (steps 2 and 3 ) is base-catalyzed by an active-site Glu residue (shown as B:). The proton (pink) initially at C-2 is made more easily abstractable by electron withdrawal by the adjacent carbonyl and the nearby hydroxyl group.

• After its transfer from C-2 to the active-site Glu residue, the proton is freely exchanged with the surrounding solution; that is, the proton abstracted from C-2 in step 2 is not necessarily the same one that is added to C-1

• Step 3 (The additional exchange of protons (yellow and blue) between the hydroxyl groups and solvent is shown for completeness. The hydroxyl groups are weak acids and can exchange protons with the surrounding water whether the isomerization reaction is underway or not.)

3. Phosphorylation of fructose 6-phosphate: the first committed

step in glycolysis

F 6-P

F 1,6-BP

• Phosphofructokinase-1 (PFK-1)-- fructose 6-phosphate to fructose 1, 6 bisphosphate. Irreversible under cellular condition.

• PFK-2: fructose 6-phosphate to fructose 2, 6 bisphosphate.

• Major regulatory enzyme in glycolysis – committed step - In some organisms, fructose 2,6-bisphosphate is a potent allosteric activator of PFK-1.

• The enzyme (PFK-1) is inhibited whenever the cell has ample ATP and is well supplied by other fuels such as fatty acids. When increases??

4. Cleavage of fructose 1,6-bisphosphate

F 1,6-BP

DHAP

G 3-P

5. Interconversion of the triose phosphate

Interconversion of the Triose phosphates— End of preparatory phase of glycolysis

6. Oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate

1,3-BPG

7. Phosphoryl transfer from 1,3-bisphosphoglycerate to ADP

3-PGA

Substrate-level phosphorylationsoluble enzymeschemical intermediates

Respiration-linked phosphorylationPhotophosphorylation

membrane-bound enzymestransmembrane gradients of protons

The formation of ATP by phosphoryl group transfer from a substrate is referred to as a

substrate-level phosphorylation

Substrate-level phosphorylation

Respiration-linked phosphorylation or Photophosphorylation

H+ H+

ATP

ADP

Glyceraldehyde 3-phosphate dehydrogenase and Phosphoglycerate kinase are coupled in

vivo

• Glyceraldehyde 3-phosphate dehydrogenase catalyzes an endergonic reaction while phosphoglycerate kinase catalyzes an exergonic reaction.

• When these two reactions are coupled (which happens in vivo), the overall reaction is exergonic.

8. Conversion of 3-phosphoglycerate to 2-

phosphoglycerate

2-PGA

The phosphoglycerate mutase reaction

The phosphoglycerate mutase reaction

3-phosphoglycerate2,3-bisphosphoglycerate2-phosphoglycerate

Phospho-glycerate mutase

COO- |HCOH | CH2O

9. Dehydration of 2-phosphoglycerate to phosphoenolpyruvate

PEP

10. Transfer of the phosphoryl group from phosphoenolpyruvate to ADP

Glucose + 2ATP + 2NAD+ + 4ADP + 2Pi

2 pyruvate + 2ADP + 2NADH + 2H+ + 4ATP + 2H2O

Glucose + 2ADP + 2NAD+ + 2Pi 2 pyruvate + 2ATP + 2NADH +

2H+

在有氧狀況下，產生的 NADH很快就被送到mitochondria中用來合成 ATP

Questions??

• The fate of the carbon skeleton of glucose

• The input of Pi and ADP and the output of ATP

• The pathway of electrons in the oxidation-reduction reactions

14.2 Feeder Pathways for Glycolysis

Feeder Pathways for Glycolysis

Glycolysis

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Phosphorolysis

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2

2

3

GlycolysisGlycolysis

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3

Glycogen and Starch Are Degraded by Phosphorolysis

• Phosphorolysis: Glycogen (animal tissues and in microorganisms) is degraded by glycogen phosphorylase (attack by Pi) at a nonreducing end to generate glucose 1P; repetitively until it approaches an (1, 6) branch point

• starch (plants) use starch phosphorylase in the same way.

phosphoglucomutase

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p.536

Branch point (16) is removed by debranching enzyme

p.536

P

P P P P

P

P

Pphosphorylase

Transferase activity ofDebranching enzyme-1,6 glucosidase activity ofDebranching enzyme

Dietary Polysaccharides and Disaccharides Undergo Hydrolysis to Monosaccharides

• Starch is the major source of carbohydrates; firstly salivary amylase breaks glycosidic bond water, not Pi, is the attacking species.

• Pancreas-amylase continues the breakdown process - maltose and maltotriose (the di- and trisaccharides of (1→4) glucose) and limit dextrins, fragments of amylopectin containing (1→6) branch points.

• Maltose and dextrins are degraded by enzymes of the intestinal brush border (the fingerlike microvilli of intestinal epithelial cells, which greatly increase the area of the intestinal surface).

2

Dietary polysaccharides and disaccharides are hydrolyzed to monosaccharides

• Lactose intolerance — lactose cannot be completely digested and absorbed in the small intestine, and in the large intestine the lactose is converted by bacteria into toxic products that cause abdominal cramps and diarrhea.

• Complication- undigested lactose and its metabolites increase the osmolarity of the intestinal contents, favoring the retention of water in the intestine.

Conversion of galactose to glucose 1-phosphate

• from the intestine to the liver - Galactose is phosphorylated at C-1 by galactokinase firstly .

• UDP galactose, which is formed when galactose 1P displaces glucose 1P from UDP-glucose by UDP-glucose; galactose 1P uridylyltransferase.

• UDP-galactose is then converted by UDP-glucose 4-epimerase to UDP-glucose, in a reaction that involves oxidation of C-4 by NAD, then reduction of C-4 by NADH; the result is inversion of the configuration at C-4.

• The UDP glucose is recycled through another round of the same reaction - conversion of galactose 1P to glucose 1P; there is no net production or consumption of UDP-galactose or UDP-glucose.

3

Glycolysis

Epimer and epimerase (p. 241)

• Two sugars that differ only in the configuration around one carbon atom are called epimers.

• Enzymes that catalyze inversion of the configuration about an asymmetric carbon in a substrate having more than one center of asymmetry are called epimerases.

Galactosemia - 半乳糖血 ( 症 )

• Galactokinase deficiency galactosemia, high galactose concentrations are found in blood and urine. Infants develop cataracts( 白內障 ), caused by deposition of the galactose metabolite Galactitol in the lens - strict limitation of galactose in the diet.

• Transferase-deficiency galactosemia - poor growth in children, speech abnormality, mental deficiency, and liver damage that may be fatal.

• Epimerase-deficiency galactosemia leads to similar symptoms

14.3 Fates of Pyruvate under Anaerobic Conditions:

Fermentation

Three possible catabolic fates of the pyruvate

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3 CH16

1. Pyruvate is the terminal electron acceptor in lactic acid fermentation

• When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of pyruvate and NADH produced in glycolysis.

• NAD+ is regenerated from NADH by the reduction of pyruvate to lactate (lactate dehydrogenase)

• The lactate formed by active skeletal muscles can be recycles; --- in blood to liver and converted to glucose (Cori Cycle)

Fermentation

• Fermentation is referring to the process when no oxygen is consumed or no change in the concentration of NAD+ or NADH during energy extraction.

• Fermentation is a general term for the anaerobic degradation of glucose or other organic nutrients to obtain energy (ATP).

p.538

2 NAD+

2 lactate2 NAD+

2 NADH 2 pyruvate

2 NADH2 pyruvate2 ATP

The purpose of fermentation is to regenerate NAD+

• In order to continue regenerating NAD+, cells come up a strategy.

• During fermentation, NAD+ is regenerated during the reduction of pyruvate, the product of glycolysis.

glucose

2 NAD+

2 ADP

glycolysis

fermentation2 lactate2 NAD+

2 NADH 2 pyruvate

2 NADH2 pyruvate2 ATPglucose

2 NAD+

2 ADP

glycolysis

fermentation2 lactate2 NAD+

2 NADH 2 pyruvate

2 NADH2 pyruvate2 ATPglucose

2 ADP

glycolysis

fermentation

2. Ethanol is the reduced product in alcohol fermentation

• Yeast and other microorganisms ferment glucose to ethanol and CO2,

• The CO2 produced by pyruvate decarboxylation in brewer’s yeast is responsible for the characteristic carbonation of champagne.

• In baking, CO2 released by pyruvate decarboxylase when yeast is mixed with a fermentable sugar causes dough to rise.

• Pyruvate decarboxylase (not oxidation); is absent in vertebrate tissues.

• Alcohol dehydrogenase (human liver) oxidation of ethanol from ingested or produced by intestinal microorganisms (in company with the reduction of NAD+ to NADH)

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2

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1

(human liver)

Thiamine pyrophosphate (TPP) is the coenzyme of pyruvate

decarboxylase

• Thiamine pyrophosphate is derived from vitamin B1 (thiamine).

• Lack of vitamine B1 will lead to beriberi (edema, pain, paralysis, death; Singhalese “I cannot” Signifying the person is too ill to do anything.).

Fermentations Yield a Variety of Common Foods and Industrial Chemicals

• Yogurt (Lactobacillus bulgaricus) – lactic acid; the resulting drop in pH; Swiss cheese (Propionibacterium freudenreichii), - propionic acid and CO2; pickles, sauerkraut, sausage, soy sauce, kimchi (Korea)…

• drop in pH associated with fermentation also helps to preserve foods.

• Clostridium acetobutyricum ferments starch to butanol and acetone.

• Industrial fermentations: These fermentations are generally carried out in huge closed vats in which temperature and access to air are adjusted to favor the multiplication of the desired microorganism and to exclude contaminating methanol, formic, acetic, propionic, butyric, and succinic acids, and glycerol, ethanol, isopropanol, butanol, and butanediol.

• Some industrial fermentations – immobilize the cells in an inert support, to pass the starting material continuously through the bed of immobilized cells, and to collect the desired product in the effluent.

Fermentation has commercial values

• Bacteria like Lactobacillus bulgaricus (yogurt) and Propionibacterium freudenreichii (swiss cheese) ferments milk to produce lactic acid or propionic acid and CO2.

Cori Cycle• The cycle of reactions that

includes glucose conversion to lactate in muscle and lactate conversion to glucose in liver is called the Cori cycle

• lactate (in the blood) is slowly converted back to glucose by gluconeogenesis (need oxygen to produces ATP) in the liver in rest or recovery period - oxygen debt.

• The circulatory systems of most small vertebrates can carry oxygen to their muscles fast enough to avoid having to use muscle glycogen anaerobically.

• The circulatory systems of larger animals, including humans, cannot completely sustain aerobic metabolism in skeletal muscles over long periods of intense muscular activity.

14.4 Gluconeogensis

Carbohydrate synthesis from simple precursors• In mammals, human brain (120

g of glucose each day)—more and nervous system, as well as the erythrocytes, testes, renal medulla, and embryonic tissues, depend almost completely on glucose for their metabolic energy.

• Gluconeogenesis (in liver and renal cortex) –precursors are lactate, pyruvate, glycerol, and glucogenic amino acids (A,

• In plant seedlings, stored fats and proteins are converted, via paths that include gluconeogenesis, to the disaccharide sucrose for transport throughout the developing plant. Glucose and its derivatives are precursors for the synthesis of plant cell walls, nucleotides, coenzymes, and metabolites.

• Many microorganisms, gluconeogenesis starts from acetate, lactate, and propionate.

[NADH] ↓

Gluconeogenesis and glycolysis are not identical pathways running in opposite directions

• seven of the ten reactions of gluconeogenesis are the reverse of glycolytic reactions. Except: (1). the conversion of glucose to glucose 6P by hexokinase; (2). the phosphorylation of fructose 6P to fructose 1,6-bisP by PFK-1, and (3). the conversion of PEP to pyruvate by pyruvate kinase.

• Both glycolysis and gluconeogenesis are irreversible processes in cells.

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in mitochondria

in cytosol

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2

in liver, kidney, not muscle and brain

Three glycolysis reactions are involved in large negative free-energy change

First bypass: Phosphorylation of pyruvate to PEP in two routes in cytosol and mitochondria

• the predominant path (use pyruvate or alanine) -Pyruvate is transported from the cytosol into mitochondria or is generated from alanine within mitochondria by transamination, (the -amino group is removed from alanine and leaving pyruvate), then Pyruvate carboxylase (coenzyme biotin) converts the pyruvate to oxaloacetate (OAA).

• the mitochondrial membrane has no OAA transporter, OAA is reduced to malate by mitochondrial malate dehydrogenase, Malate leaves the mitochondrion through a malate transporter (inner mito mm.) and is reoxidized to OAA.

• In the cytosol, OAA is converted to PEP by PEP carboxykinase.

• 2 ATPs are required for (pyruvate to PEP).

• CO2 added to pyruvate in the pyruvate carboxylase step is the same molecule that is lost in the PEP carboxykinase reaction (activation)

• second pathway, (lactate) as precursor.

in mitochondria

(OAA)

in cytosol

via. malate transporter

deamination of alanine

lactate

1

Alternative paths from pyruvate to PEP

• Conversion of lactate to pyruvate in the cytosol of hepatocytes yields NADH - no need to export reducing equivalents.

• Pyruvate in mitochondria is converted to OAA - converted directly to PEP by a mitochondrial isozyme of PEP carboxykinase, and the PEP is transported out of the mitochondrion to continue on the gluconeogenic path.

• The mitochondrial and cytosolic isozymes of PEP carboxykinase are encoded by separate genes in the nuclear chromosomes, providing another example of two distinct enzymes catalyzing the same reaction but having different cellular locations or metabolic roles (i.e. isozymes of hexokinase).

[NADH]↑

Second Bypass: Conversion of Fructose 1,6 - BisP to Fructose 6P

• the generation of fructose 6-phosphate from fructose 1,6-bisphosphate is catalyzed Mg 2+ -dependent fructose 1,6- bisphosphatase (FBPase-1), which promotes the hydrolysis of the C-1 phosphate.

2

Third Bypass: Conversion of Glucose 6P to Glucose

• catalyzed by glucose 6-phosphatase – Mg 2+ -activated enzyme is found on the lumenal side of the endoplasmic reticulum (ER) of hepatocytes and renal cells

• Muscle and brain tissue do not contain glucose 6-phosphatase and so cannot carry out gluconeogenesis.

• Glucose produced by gluconeogenesis in the liver or kidney or ingested in the diet is delivered to brain and muscle through the bloodstream.

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Gluconeogenesis Is Energetically Expensive but Essential

• Citric Acid cycle (TCA) intermediates (isocitrate, -ketoglutarate, succinyl-CoA, succinate, fumarate, and malate → OAA.

• Glucogenic amino acid: Some or all of the carbon atoms of most amino acids derived from proteins are ultimately catabolized to pyruvate or to intermediates of the TCA. Such amino acids can convert to glucose.

• no net conversion of fatty acids to glucose occurs in mammals - pyruvate deHase reaction is irreversible (No acetyl-CoA to pyruvate). Plants, yeast, and bacteria use glyoxylate cycle (converting acetyl-CoA to OAA).

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

• How many ATP, GTP, and NADH is required when formation of one molecule of glucose from pyruvate??

14.5 Pentose Phosphate Pathway of Glucose Oxidation

General scheme of the Pentose Phosphate Pathway

• Rapidly dividing cells, such as those of bone marrow, skin, and intestinal mucosa, use the pentoses to make RNA, DNA, and such coenzymes as ATP, NADH, FADH2, and coenzyme A.

• Liver, adipose, lactating mammary gland, erythrocytes - the electron donor NADPH, needed for reductive biosynthesis or to counter the damaging effects of oxygen radicals.

• By maintaining a reducing atmosphere (a high value of [NADPH]/[NADP] and a high [GSH]/[GSSG], GS = glutathione), - prevent or undo oxidative damage to proteins, lipids, and other sensitive molecules.

Rapidly dividing cells, such as those of bone marrow, skin, and intestinal mucosa,

Oxidative pentose phosphate pathway

• Produces 1). NADPH – carries chemical energy in the form of reducing power and is used universally as the reductant in anabolic pathway (fatty acids and steroids); 2). ribose 5-phosphate- biosynthesis of nucleic acids; and 3). CO2

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Favism-蠶豆症

• 25% occur in tropical Africa, parts of the Middle East, and Southeast Asia, areas where malaria is most prevalent. Studies show that growth of malaria parasite, Plasmodium falciparum, is inhibited in G6PD-deficient erythrocytes. The parasite is very sensitive to oxidative damage and is killed by a level of oxidative stress that is tolerable to a G6PD-deficient human host. Because the advantage of resistance to malaria balances the disadvantage of lowered resistance to oxidative damage, natural selection sustains the G6PD-deficient genotype in human populations where malaria is prevalent.

• Affected 400 million - asymptomatic• erythrocytes are lysed within 24 to

48 hours after ingestion of the fava beans ( 蠶豆 ), - Hb into the blood. Jaundice and kidney failure. Similar symptoms can occur with ingestion of the antimalarial drug primaquine or of sulfa antibiotics or following exposure to certain herbicides.

• primaquine and fava beans (divicine) – H2O2 and superoxide radical.

• detoxification, H2O2 is converted to H2O by reduced glutathione and glutathione peroxidase (or catalase), and the oxidized glutathione is converted back to the reduced form by glutathione reductase and NADPH.

Ribulose 5-phosphate is epimerized to xylulose 5-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

Nonoxidative PPP – recycling Glucose 6P

• 6 (5C) to 5 (6C)

Glucose 6P

Dihydroxyacetone P

Oxidative PPP

Transketolase

• Transketolase catalyzes the transfer of a two-carbon fragment from a ketose donor to an aldose acceptor.

• TPP (thiamine pyrophosphate- vit. B1) is needed as cofactor

Transaldolase

• transaldolase similar to the aldolase reaction of glycolysis: a three-carbon fragment is removed from sedoheptulose 7-phosphate and condensed with glyceraldehyde 3-phosphate, fructose 6-phosphate and erythrose 4-phosphate.

Oxidative PPP Vs. Nonoxidative PPP

• Oxidative PPP is irreversible; nonoxidative PPP is reversible – converting hexose phosphates to pentose phosphates (reductive pentose phosphate pathway of plants – photosynthetic assimilation of CO2)

• All in cytosol- glycolysis, gluconeogenesis, and PPP are connected (the cell’s relative needs for pentose phosphates, NADPH, and ATP)

• Wernicke-Korsakoff Syndrome (severe memory loss, mental confusion, and partial paralysis) Is exacerbated (使惡化 ) by a Defect in Transketolase (1/10 affinity for TPP). The syndrome is more common among alcoholics (chronic alcohol consumption interferes with the intestinal absorption of some vitamins, including thiamine).

Glucose 6-Phosphate Is Partitioned between Glycolysis and the Pentose Phosphate Pathway

• When a cell is rapidly converting NADPH to NADP in biosynthetic reductions, the level of NADP rises, allosterically stimulating G6PD and thereby increasing the flux of glucose 6-phosphate through the pentose phosphate pathway.

• When NADPH is forming faster than it is being used for biosynthesis and glutathione reduction , [NADPH] rises and inhibits the first enzyme in the pentose phosphate pathway. As a result, more glucose 6-phosphate is available for glycolysis.

• Quiz next week