CARBOHYDRATE CARBOHYDRATE METABOLISMMETABOLISM

Week 3-5

Major pathways of glucose utilizationMajor pathways of glucose utilization

LIVER BLOOD MUSCLE

glycogen glycogen

Fructose & galactose

glucose glucose Glucose

ATP ATP ATP

pyruvate pyruvate pyruvate

Lactate Lactate ATP

CO2 + H2O CO2 + H2O

Lipid

sterol & cholesterol

glucose has a normal blood level of 70-90 mg/dL< 70 mg/ 100 ml hypoglycemia> 70 mg/ 100 ml hyperglycemia

glicolysisglicolysisGlycolysis is a series of reactions that takes place in the

cytoplasm of all prokaryotes and eukaryotesa molecule of glucose is degraded in a series of enzyme-

catalyzed reactions to yield two molecules of the three-carbon compound pyruvate

Also called Embden Mayerhoff metabolismAerobic (glucose pyruvate) and anaerobic (glucose

lactate)Fermentation (glucose ethanol)

Three possible catabolic fates of the pyruvate formed in Three possible catabolic fates of the pyruvate formed in glycolysis.glycolysis.

An Overview: Glycolysis Has Two PhasesAn Overview: Glycolysis Has Two Phases

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.

Entry of glycogen, starch, disaccharides, and hexoses into the preparatory stage of glycolysis

Fates of Pyruvate under Anaerobic Conditions: Fermentation

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.

Pyruvate is the terminal electron acceptor in Lactic Acid Pyruvate is the terminal electron acceptor in Lactic Acid FermentationFermentation

Under anaerobic or hypoxic conditions, many organisms regenerate NAD by transferring electrons from NADH to pyruvate, forming lactate

Ethanol Is the Reduced Product in Ethanol Fermentation

Other organisms, such as yeast, regenerate NAD by reducing pyruvate to ethanol and CO2.

In these anaerobic processes (fermentations), there is no net oxidation or reduction of the carbons of glucose.

GluconeogenesisGluconeogenesis

Gluconeogenesis is the pathway for glucose synthesis from noncarbohydrate precursors

Important for the maintenance of blood glucose levels during starvation or during vigorous exercise.

The brain and erythrocytes depend almost entirely on blood glucose as an energy source.

Gluconeogenesis occurs mainly in the liver and to a lesser extent in the kidney.

Most enzymes of gluconeogenesis are cytosolic, but pyruvate carboxylase and glucose 6-phosphatase are located in the mitochondrial matrix and bound to the smooth endoplasmic reticulum, respectively.

The Pathway of GluconeogenesisThe Pathway of Gluconeogenesis

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

Three irreversible steps in the glycolytic pathway are bypassed by reactions catalyzed by gluconeogenic enzymes: ◦ conversion of pyruvate to PEP via

oxaloacetate, catalyzed by pyruvate carboxylase and PEP carboxykinase;

◦ dephosphorylation of fructose 1,6-bisphosphate by FBPase-1; and

◦ dephosphorylation of glucose 6-phosphate by glucose 6-phosphatase.

Alternative paths from pyruvate to phosphoenolpyruvate

Conversion of mitochondrial pyruvate to cytosolic phosphoenolpyruvate to initiate gluconeogenesis.

Oxaloacetate cannot pass across the inner mitochondrial membrane, so it is reduced to malate, which can do so.

The initial irreversible step of glycolysis is bypassed by glucose 6-phosphatase, which catalyzes the dephosphorylation of glucose 6-phosphate to form glucose◦ This enzyme is mainly found in liver and kidney, the only two

organs capable of releasing free glucose into the blood.◦ A special transporter (GLUT2) in the membranes of these

organs allows release of the glucose.

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

The Cori CycleThe Cori Cycle

During vigorous exercise, pyruvate produced by glycolysis in muscle is converted to lactate by lactate dehydrogenase.

The lactate diffuses into the bloodstream and is carried to the liver. Here it is converted to glucose by gluconeogenesis. The glucose is

released into the bloodstream and becomes available for uptake by muscle (as well as other tissues, including brain).

Pentose Phosphate PatwayPentose Phosphate PatwayThe pentose phosphate pathway (PPP), also called

the hexose monophosphate shunt, is an alternate pathway of glucose metabolism that supplies the NADPH required by many biosynthetic pathways.◦ The main purpose of the PPP is to generate NADPH to be

used in pathways for synthesis of important molecules, eg, amino acids, lipids, and nucleotides.

◦ NADPH derived from the PPP is also important for detoxification of reactive oxygen species.◦ The PPP also is responsible for synthesis of ribose 5-

phosphate for nucleotide biosynthesis

The PPP operates in two phases: an oxidative phase and a nonoxidative phase

Glycogen MetabolismGlycogen MetabolismGlycogen is stored in muscle and liver as

large particles. Contained within the particles are the

enzymes that metabolize glycogen, as well as regulatory enzymes

Glycogen granules in a hepatocyte

glycogenolysisglycogenolysis

the catabolic pathways from glycogen to glucose 6-phosphate Catalyzed by glycogen phosphorylase

Glycogen synthesisGlycogen synthesis

A glycogen chain is elongated by glycogen synthase. The enzyme transfers the glucose residue of UDP-glucose to the nonreducing end of a glycogen branch to make a new (α-14) linkage

Branch synthesis in glycogen

The glycogen-branching enzyme (also called amylo (14) to (16) transglycosylase or glycosyl-(46)-transferase) forms a new branch point during glycogen synthesis

Glycogen degradation and glycogen synthesis are reciprocally regulated by hormones.

Kinases and phosphatases control the activities of the interconvertible enzymes glycogen phosphorylase and glycogen synthase

THE CITRIC ACID THE CITRIC ACID CYCLECYCLE

Hans Krebs (1900–1981). Krebs wasawarded the Nobel Prize in Physiology orMedicine in 1953 for his discovery of the citricacid cycle.

Catabolism of proteins, fats, and Catabolism of proteins, fats, and carbohydrates in the three stages carbohydrates in the three stages of cellular respirationof cellular respiration

Production of Acetyl-CoA (Activated Acetate)Production of Acetyl-CoA (Activated Acetate)

Pyruvate, the product of glycolysis, is converted to acetyl-CoA, the starting material for the citric acid cycle, by the pyruvate dehydrogenase complex.

Coenzyme ACoenzyme A

Oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex

Reactions of the citric acid cycleReactions of the citric acid cycle

Products of one turn of the citric acid cycleProducts of one turn of the citric acid cycle

Biosynthetic precursors produced by an incompleteBiosynthetic precursors produced by an incompletecitric acid cycle in anaerobic bacteriacitric acid cycle in anaerobic bacteria

Role of the citric acid cycle in anabolismRole of the citric acid cycle in anabolism

Regulation of the Citric Acid CycleRegulation of the Citric Acid Cycle

THE GLYOXYLATE THE GLYOXYLATE CYCLECYCLE

The glyoxylate cycle is active in the germinating seeds of some plants and in certain microorganisms that can live on acetate as the sole carbon source.

In plants, the pathway takes place in glyoxysomes in seedlings.

It involves several citric acid cycle enzymes and two additional enzymes: isocitrate lyase and malate synthase.

Reaction of glyoxylate cycle

Relationship between the glyoxylate and citric acid cycles

Coordinated regulation of glyoxylate and citric acid cycle

ELECTRON TRANSPORT CHAIN (ETC)/ RESPIRATION

Most energy from food obtained through stepwise anaerobic oxidative processes to yield NADH or FADH2 (reducing equivalent).

ThenNADH or FADH2 aerobically oxidized ( in ETC ).This energy is used to synthesize ATP

(phosphorylation).

But how the energy of ETC

(oxidation) is used to

synthesize ATP (phosphorylation)

The coupling of oxidation & phosphorylation.

Peter Mitchell

Chemiosmotic Theory

A proton gradient is generated with energy from electron transport by the vectorial transport of protons (protonpumping) by Complexes I, III, IV from the matrix tointermembrane space of the mitochondrion.

Mitochondrion or the power house of cell

• Outer membrane permeable to small molecules

• Inner membraneImpermeable to small molecules.

• Cristae increase areaIT contains:

Electron transport system (ETC) and ATP synthase complex embedded;

• Integrity required for coupling ETC to ATP synthesis• Matrix contains Krebs cycle enzymes, β-oxidation enzymes;

also ATP, ADP, NAD, NADH2, Mg2+, etc

The size : (1-2μ)The number: 1-1000s in each cell

Chemiosmotic TheoryChemiosmotic Theory

Ubiquinone and cytochrome c are mobile carriers. They ferry electrons from one complex to the next

ETC= electron transport chain

• NADH dehydrogenase (NADH Q reductase)

• Huge protein– 25 pp

• FMN, Fe-S• Electron UQ

• NADH dehydrogenase (NADH Q reductase)

• Huge protein– 25 pp

• FMN, Fe-S• Electron UQ

Iron-Sulfur Centers Transfer of electrons in variety of proteins such as NADH and

succinate dehydrogenase

2H+ + 2 e-

Coenzyme Q

Coenzyme Q

Coenzyme Q = Ubiquinonea lipid in inner membrane carries electrons polyisoprene tail moves freely within membrane

Complex II: Succinate Q Recuctase (Succinate dehydrogenase)Is the only membrane bound enzyme in the TCA cylce and contains FAD, Fe-S

II electrons UQ

Complex III= Cyt C reductase

Complex IV (Cytochrome C oxidase)

Heme A and Cu act together totransfer electrons to oxygen

e- from cyt c to a

Cyto oxidaseContains a, a3, and CuA, CuB

The detail of this electron transfer in Complex IV is not knownIt also functions as a proton pump

Cu(II) Cu(I)

Membrane potential = 140 mVpH gradient = 60 mVTotal proton motive force = 200 mV

ATP Synthase (F0 - F1 complex)

F0

FI

F0 = Oligomycin sensitive Fragment

ATP synthesis at FATP synthesis at F11 results from results fromrepetitive comformational changesrepetitive comformational changesas as γγ rotates rotates

Uncoupling ProteinUncoupling ProteinThe coupling of oxidation (to make Proton gradient) and phosphorylation ( ADP+P) is needed for ATP synthesis.*Thermogenin is a proton carrier located at inner mitochondrial membrane*

*This uncoupling protein produced in brown adipose tissue of newborn mammals, and hibernating mammals for cold adaptation.

Uncoupling ProteinUncoupling ProteinThe uncoupling protein blocks development of a H+ electrochemical gradient, thereby stimulating respiration. ∆G of respiration is dissipated as heat.

This "non-shivering thermogenesis" is costly in terms of respiratory energy unavailable for ATP synthesis, but provides valuable warming of the organism.

The gene is activated by thyroid hormone

Different level of the hormone in different season and areas

Poisons of Oxidative Poisons of Oxidative PhosphorylationPhosphorylation

1- OXIDATION (ETC) inhibitors.2- PHOSPHORYLATION inhbitors.3- Uncouplers.4- ATP/ADP transporter

(tanslocators) inhibitors

Rotenone, amytalAntimycin A

Dimercaprol

HCN, CO, H2S

• Complex 1: Rotenone and Barbiturates such as amobarbital and amytal inhibit NDAH- DH. They are fatal at sufficient dosage.

• 2- Complex 2: Malonate is competitive inhibitor of Suc- DH• 2- Complex 3: Antimycin A and Dimercaprol inhibit cyt C reductase.• 3- Complex 4: Classic poisons HCN, CO, H2S arrest respiration by inhibiting

cyt oxidase.• Note: all the components of the respiratory chain before the block

become reduced, all the components• downstream become oxidized.

ETC inhibitorsETC inhibitors

ATP Synthase and ATP/ADP ATP Synthase and ATP/ADP translocator inhibitors translocator inhibitors

The antibiotic Oligomycin completely blocks F0 ( Oligomycin sensitive Fragment) the flow of H+ through the F0 directly inhibiting ox-phos.

Atractyloside ATP/ADP translocator.

Uncouplers are lipid-soluble weak acids. E.g., H+ can dissociate from the OH group of the uncoupler dinitrophenol.

Uncouplers dissolve in the membrane and function as carriers for H+.

OH

NO2

NO2

2,4-dinitrophenol

Uncoupling reagents (uncouplers)

It is very efficient processIt is very efficient process•Recall living cells efficiency is ~ 42%, compared to about 3% efficiency when burning oil or gasoline. BUT HOW?Separating carbohydrates, lipids, etc. from oxygen to optimize recover of energy. In other words first they are anaerobically oxidized to yield NADH and FADH2,And then Stepwise aerobic oxidation of NADH and FADH2 through ETC And then ATP synthesis by electrochemical energy.

How is the energy yield in living How is the energy yield in living cells cells

SummarySummary1. Oxidative Phosphorylation is carried out by

respiratory assemblies that are located in the inner membrane...

2. Respiratory assemblies contain numerous electron carriers, Such as cytochromes.

3. When electrons are transferred, H+ are pumped out.

4. ATP is formed when H+ flow back to the mitochondria.

5. Oxidation and phosphorylation are COUPLED

6. The oxidation of NADH 3 ATP, and FADH2 2 ATP