洪錦堂 老師 醫學大樓 0866 室 講義在我個人網站 ( 生化科 )

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洪錦堂 老師醫學大樓 0866 室

講義在我個人網站 ( 生化科 )

• Chapter 14 Glycolysis, gluconeogenesis, and the pentose phosphate pathway

• Chapter 15 Principles of metabolic regulation: Glucose and glycogen

• Chapter 16 The citric acid cycle

• Chapter 19 Oxidative phosphorylation

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To remind you…..

• Class schedule

• Exam

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GlycolysisGluconeogenesis

Pentose phosphate pathway

Chapter 14

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• Catabolic pathways converge to a few end products

• Anabolic pathways diverge to synthesize many biomolecules

• Some pathways serve both in catabolism and anabolism, such pathways are amphibolic

Metabolism: Catabolism & Anabolism

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• 14.1 Glycolysis

• First Phase of Glycolysis (preparatory phase)

• Second Phase of Glycolysis (lysis phase)

• 14.2 Feeder pathways for glycolysis

• 14.3 Fates of pyruvate under anaerobic conditions: Fermentation

• 14.4 Glucogenesis

• 14.5 Pentose phosphate pathway of glucose oxidation

Outline

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14.1 Glycolysis

Why glucose?1. Complete oxidation yields 2,840 KJ/mol2. low cytosolic osmolarity3. capable of supplying a huge array of metabolic intermediates for biosynthetic reactions

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Glycolysis又稱 The Embden-Meyerhof (Warburg) Pathway

1. Consume 2 ATP in 1st phase, produce 4 ATP in 2nd phase: net gain 4 - 2 = 2 ATP

2. Glucose(6C) 2 ＊3C (pyrurate)

3. Pyruvate is used in 3 ways:

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Alcohol fermentation

Lactate fermentation

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Hexokinase

Phosphohexoseisomerase

Phospho-Fructosekinase-1

Aldolase

Triose phosphate isomerase

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Glyceraldehyde 3-phosphateDehydrogenase

Phospho-GlycerateKinase

Phospho-GlycerateMutase

Enolase

Pyruvatekinase

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Glycolytic enzyme 命名(1) Kinase 與 ATP 水解 or 合成相關之 enzyme

(2) Mutase( 轉換 )Transfer functional group from one position to another in the same molecule, 指～ P 由 C3 位置 C2位置

(3) Isomerase 指 Aldose Ketose

(4) Aldolase 指產物各為 -Aldose 分子及 ketose 分子

(5) Enolase 產生 enol form 分子

-C=C- alcohol

※Mutase is a subclass of isomerase

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The first reaction - phosphorylation of glucose

• Hexokinase or glucokinase

• This is a priming reaction - ATP is consumed here in order to get more later

• ATP makes the phosphorylation of glucose spontaneous

First Phase of Glycolysis

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Reaction 1: Phosphorylation of glucose

spontaneous and irreversible

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Hexokinase is allosterically inhibited by its reaction product

• Hexokinase:• Km 0.1 mM• So hexokinase is nor

mally active! • Inhibited by G6P rever

sibly• Glucose, mannose….

• Glucokinase:• Km 10 mM• only turns on when ce

ll is rich in glucose• Not inhibited by G6P• Exist in liver• Specific for glucose

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Irreversible

Reaction 2: Phosphohexose isomerase (phosphoglucose isomerase)

Reaction 3: Phosphofructose kinase (PFK-1)---- 2nd priming step

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Reaction 4: Fructose 1,6-bisphosphate Aldolase

In erythrocyte –0.23kJ/mol

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Reaction 5: DHAP→Glyceraldehyde 3-P by triose-phosphate isomerase

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Glycolysis - Second Phase

Metabolic energy produces 4 ATP

• Net ATP yield for glycolysis is two ATP

• Second phase involves two very high energy phosphate intermediates

• .

– 1,3 BPG

– Phosphoenolpyruvate

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Reaction 6: Glyceraldehyde-3-phosphate dehydrogenase

1.Addition of a phosphate group to G3P

2.e- transfer from G3P to NAD+ (hydride H:-)

3.No ATP or ADP is involved

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Reaction 7: Phosphoryl transfer from 1,3 bisphosphoglycerate to ADP

Pay off

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The formation of ATP by phosphoryl transfer from a substrate such as 1,3 BPG is referred as a substrate-level phosphorylation. It involves soluble proteins and chemical intermediates.

Respiration-linked (oxidative) phosphorylation( 氧化磷酸化 ) involves membrane-bound enzymes and transmembrane gradients of protons.

Substrate-level phosphorylation

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Reaction 8: Conversion of 3-phosphoglycerate to 2 phosphoglycerate

Reaction 9: Dehydration of 3- to 2- phosphoenolpyruvate by enolase( 烯醇酉每 )

高能量

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Payoff

Reaction 10: Transfer of the phosphoryl group from phosphoenolpyruvate to ADP

This is referred to as "substrate-level phosphorylation

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SUMMARY 14.1 Glycolysis

1. 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.

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

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SUMMARY 14.1 Glycolysis

3. 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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SUMMARY 14.1 Glycolysis

4. 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 formation of one NADH and two ATP per triose phosphate oxidized. The net equation for the overall process is

Glucose+2NAD++2ADP+2Pi 2pyruvate+2NADH+2H+ +2ATP+2H2O

4e-

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5. Glycolysis is tightly regulated in coordination with other energy-yielding pathways to assure a steady supply of ATP. Hexokinase, PFK-1, and pyruvate kinase are all subject to allosteric regulation that controls the flow of carbon through the pathway and maintains constant levels of metabolic intermediates.

SUMMARY 14.1 Glycolysis

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Glycogen and starch

Maltose, lactose, trehalose, sucrose

Fructose, mannose, galactose

14.2 Feeder pathways for glycolysis

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Fig. 14-11Conversion of galactose to glucose 1-p

hosphate

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Mutation SymptomsGalactokinase- deficient (high galactose conc. is found in the blood and urine)

Cataracts cause by deposition of galactose metabolite galactitol in the lens

Transferase-deficient

Poor growth of children, speech abnormality, mental deficiency, and liver damage even when galactose is withheld from the diet(more severe!)

Epimerase-deficient

Same as above but is less severe when dietary galactose is carefully controlled.

Galactosemia

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Catabolism of glycogen

• Cellular glycogen

• Dietary glycogen

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Glycogen phosphorylase catalyzes 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.

14-10

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

Glycolysis Pentose phosphate pathway

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Dietary polysaccharides and disaccharides are hydrolyzed to monosaccharides by various enzymes

-amylase:Salivary---digest to short polysaccharide fragment

or oligosaccharides

Pancrease---digest to disaccharides (maltose), trisaccharides (maltotrioses) and dextrins

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Intestine secrects enzymes to digest dextrin and disaccharides

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Catabolism of dietary and cellular glycogen

Cellular Dietary (saliva) Dietary (pancrease)

Enzyme Glycogen phosphorylase and debranching enzyme

-amylases -amylases

End product

glucose-1-P Short polysaccharides fragment or oligosaccharides

Di-saccharides and dextrins

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Lactose intolerance: 乳糖不耐症Lack lactase

Diarrhea and discomfort

Undigested lactose and its metabolites increase the osmolarity of the intestinal contents, favoring the retention of water in the intestine.

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( 蛀牙 )

Sucrose Plaque (polysaccharide)

Products of anaerobic glycolysis carried out by bacteria

lactate and pyruvate

tooth decay

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SUMMARY 14.2 Feeder Pathways for Glycolysis

1. Glycogen and starch, polymeric 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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SUMMARY 14.2 Feeder Pathways for Glycolysis

2. 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.

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3. A variety of D-hexoses, including fructose, galactose, and mannose, can be funneled into glycolysis. Each is phosphorylated and converted to either glucose 6-phosphate or fructose 6-phosphate.

SUMMARY 14.2 Feeder Pathways for Glycolysis

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SUMMARY 14.2 Feeder Pathways for Glycolysis

4. Conversion of galactose 1-phosphate to glucose 1-phosphate involves two nucleotide derivatives: UDP-galactose and UDP-glucose. Genetic defects in any of the three enzymes that catalyze conversion of galactose to glucose 1-phosphate result in galactosemias of varying severity.

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14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation

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yeast muscle乙醛

Fetal Alcohol syndrome

Anaerobic pathways

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• Thiamine pyrophosphate (TPP), a coenzyme derived from vitamin B1.

• Lack of vitamin B1 in the human diet leads to the condition known as beriberi.

• Thiamine pyrophosphate plays an important role in the cleavage of bonds adjacent to a carbonyl group, such as the decarboxylation of -keto acids, and in chemical rearrangements in which an activated acetaldehyde group is transferred from one carbon atom to another

Thiamine Pyrophosphate Carries “Active Acetaldehyde” Groups

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Thiamine Pyrophosphate Carries “Active Acetaldehyde” Groups

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Replenishment of NAD+

1. under anaerobic condition : Lactic acid fermentation

2. under anaerobic condition : Alcoholic fermentation

3. under aerobic condition : Mitochondria oxidation of each NADH to yield 2.5

(1.5) ATP

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Oxygen debt

Cori cycle

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SUMMARY 14.3 Fates of Pyruvate under anaerobic conditio

ns: Fermentation

1. The NADH formed in glycolysis must be recycled to regenerate NAD, which is required as an electron acceptor in the first step of the payoff phase. Under aerobic conditions, electrons pass from NADH to O2 in mitochondrial respiration.

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SUMMARY 14.3 Fates of Pyruvate under anaerobic conditions:

Fermentation

2. Under anaerobic or hypoxic conditions, many organisms regenerate NAD by transferring electrons from NADH to pyruvate, forming lactate. Other organisms, such as yeast, regenerate NAD by reducing pyruvate to ethanol and CO2.

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Cellular energy level and intermediate concentration

Glycogen phosphorylase, hexokinase, phosphofructokinase, pyruvate kinase

1. At branching point2. Large and negative free energy

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Regulation of PFK-1

FIGURE 15–18 Phosphofructokinase-1 (PFK-1) and its regulation.

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為何 ATP 對 PFK-1 是受質也是 inhibitor?

當 [ATP] 低 , 即 [ADP] 高 Low energy chargeglycolysis

當 [ATP] 高 , 即 [ADP] 低 High energy chargeglycolysis

*[ATP] 低時 ,ADP 進入 allosteric site

當 activator, ATP 進入 active site 當

substrate

*[ATP] 高時 ,ATP 進入 allosteric site

當 inhibitor

+

-

FIGURE 6–26 Subunit interactions in an allosteric enzyme, and interactionswith inhibitors and activators.

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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

14.4 gluconeogenesis

57Fig. 6-29

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• Substrates: pyruvate, lactate, glycerol, amino acids (except Lys and Leu)

• Active in liver and kidney: supply glucose to brain and muscle

• Seven of ten glycolytic steps in reverse• Three steps not reversal of glycolysis for tw

o reasons– energetics– regulation

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• Occurs mainly in liver and kidneys • Not the mere reversal of glycolysis for 2 r

easons:• Energetics must change to make glucone

ogenesis favorable (delta G of glycolysis = -74 kJ/mol

• Reciprocal regulation must turn one on and the other off - this requires something new!

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Something Borrowed, Something New

• 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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GluconeogenesisGlycolysis

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Three unique gluconeogenesis steps: First bypass

• Pyruvate to PEP– Pyruvate carboxylase converts pyruvate to oxaloac

etate• Biotin-dependent enzyme• ATP + bicarbonate to carbonylphosphate interm

ediate to carboxybiotin intermediate• Allosteric activation by acetyl CoA• Localized in mitochondria

– Oxaloacetate to malate and back (if PEP carboxykinase is localized in cytosol)

– PEP carboxykinase converts oxaloacetate to PEP• Decarboxylation drives reactions• GTP or ATP supplies Pi and drives reaction• Localized in mitochondria or in cytosol

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Synthesis of PEP from pyruvate

Overall reaction:Pyruvate + ATP + GTP + HCO3- →phosphenolpyruvate + ADP + GDP + Pi + CO2

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PEP carboxykinase-Rabbit liver : mitochondriaRat liver : cytosolicHuman liver : both

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Three unique gluconeogenesis steps: Second bypass

• Fructose-1,6 bisphosphate to fructose 6 phosphate

– Hydrolysis

– Fructose-1,6-bisphosphatase

• Acetyl CoA or citrate stimulates

• fructose-2,6-bisphosphate inhibits

• AMP inhibits

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Three unique gluconeogenesis steps: Third bypass

• Glucose-6-phosphate to glucose– Hydrolysis– Glucose-6-phosphatase

• Release product into ER lumen• Enzyme expressed in liver and

kidney (not in muscle or brain)• High Km for G6P: substrate-level

control

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• FIGURE 15–6 Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase of the ER. The catalytic site of glucose 6-phosphatase faces the lumen of the ER. A glucose 6-phosphate (G6P) transporter (T1) carries the substrate from the cytosol to the lumen, and the products glucose and Pi pass to the cytosol on specific transporters (T2 and T3). Glucose leaves the cell via the GLUT2 transporter in the plasma membrane.

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Pentose phosphate pathway of glucose oxidation

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14.5 Pentose Phosphate Pathway

aka phosphogluconate pathway, 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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Oxidative Steps of the Pentose Phosphate Pathway

• Glucose-6-P Dehydrogenase – Irreversible 1st step - highly regulated!

• Gluconolactonase – Uncatalyzed reaction happens too

• 6-Phosphogluconate Dehydrogenase– An oxidative decarboxylation (in that order!)

• Phosphopentose isomerase – converts ketose to aldose

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The Nonoxidative Steps • Phosphopentose Epimerase

– epimerizes at C-3

• Transketolase (TPP-dependent)– transfer of two-carbon units

• Transaldolase (Schiff base mechanism)– transfers a three-carbon unit

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蠶豆症

7404172005

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Clinical aspects of PPP-- 蠶豆症

• G6P dehydrogenase deficiency • G-S-S-G + NADPH + H+ → 2G-SH + NADP+ (by glu

tathione reductase)• 2G-SH + H2O2 → G-S-S-G + 2H2O (by gluthathion

e peroxidase)• increase rate of oxidation of hemoglobin• Susceptible to oxidants (antimalarial primaquine, a

spirin, or sulfonamides), or fava beans

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SUMMARY 14.5 Pentose Phosphate Pathway of

Glucose Oxidation

1. 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

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SUMMARY 14.5 Pentose Phosphate Pathway of

Glucose Oxidation

2. The first phase of the pentose phosphate pathway consists of two oxidations that convert glucose 6-phosphate to ribulose 5-phosphate and reduce NADP to NADPH. The second phase comprises nonoxidative steps that convert pentose phosphates to glucose 6-phosphate, which begins the cycle again.

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SUMMARY 14.5 Pentose Phosphate Pathway of

Glucose Oxidation

3. 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.