Metabolism

-       A process in organisms to liberate and store energy from foods and create complex molecules from simpler ones through enzyme-catalyzed reactions of both matter and energy.

-       Metabolic pathway: sequence of enzymatic reactions.

o   Product of one reaction becomes the substrate of the next reaction and the products being known as metabolites (metabolic intermediates).

Two aspects of metabolism:

Ø  Catabolism: degradative process where complex molecules are broken down into simpler ones.

Ø  Anabolism: Biosynthesis of metabolism, building block compounds into more complex molecules.  require energy through ATP!

o   Catabolism of carbohydrates begins in the mouth where starch is degradative all the way through the digestive tract until monosaccharides are produces.

o   Glucose gets broken down further by enzymes of the glycolytic pathway to produce pyruvate (or lactate).

Glycolysis

-       Major pathway of utilization of glucose in the cytosol.

-       Breakdown of glucose to provide energy + intermediates for other metabolic pathways.

o   Aerobic: 10 reactions  pyruvate

o   Anaerobic: 11 reactions  lactate

a.     Energy investment reactions

b.    Energy recovery reactions

Transport and activation of glucose.

1.     Na+-dependent monosaccharide cotransport system (for D-glucose and D-galactose)

2.     Na+-independent system (D-fructose facilitated diffusion)

1.     Phosphorylation of glucose.

o   4 isoenzymes of hexokinase are known (I-IV) where IV (glucokinase) is found in hepatocytes.

o   Most hexokinases have low Km for glucose  but glucokinase is specific for glucose and has high Kmwhich enables it to handle the high concentration of glucose in portal venous blood after a meal.

o   Glucose-6-phosphate is important for several pathways:

                                               i.     Glycolysis

                                             ii.     Glyconegenesis

                                           iii.     Glycogenolysis

                                            iv.     Glycogenesis

                                             v.     Pentose phosphate pathway

2.     Isomerization of G-6-P.

o   Converted into fructose-6-phosphate by phosphoglucose isomerase (reversible reaction).

3.     Phosphorylation of F-6-P.

o   Second ATP used to convert into fructose 1,6-bisphosphate by phosphofructokinase-1 (non-reversible reaction).

                                               i.     PFK-1 is rate-limiting enzyme for glycolysis.

4.     Cleavage of F-1,6-bisP.

o   Hydrolyzed into two 3-carbon products (dihydroxyacetone phosphate, DHAP, and glyceraldehyde 3-phosphate, G3P) by aldolase.

5.     Isomerization of triose phosphates.

o   Reversible reaction but G3P is used up in other reaction which shift equilibrium in direction of product which results in usage of all DHAP.

o   Done by triosephosphate isomerase.

6.     Oxidation of glyceraldehyde 3-phosphate (GAP).

o   Glyceraldehyde-3-P dehydrogenase catalyzes NAD+ dependent oxidation to 1,3-bisphosphoglycerate and NADH.

                                               i.     Substrate-level phosphorylation reaction

o   1,3-bisphosphoglycerate is high energy phosphate.

7.     Formation of ATP from 1,3-bisphosphoglycerate and ADP.

o   1,3-BPG is used to form ATP + 3-phosphoglycerate by phosphoglycerate kinase.

§  Only reaction involving ATP and is reversible under normal cell conditions.

§  Substrate level phosphorylation

o   2 molecules of triose phosphate are formed per 1 molecule of glucose  2 ATP molecules are formed at this stage.

8.     Isomerisation of 3-P-glycerate to 2-p-glycerate.

o   Remaining part of glycolysis is aimed to convert low energy phosphor-acyl-ester of 3PG into high energy 2PG by phosphoglycerate mutase.

9.     Dehydration of 2-P-glycerate.

o   Converted to phosphoenoylpyruvate with high-energy enol phosphate by enolase.

10.  Formation of pyruvate.

o   Strong exergonic reaction  high energy release from conversion to pyruvate by pyruvate kinase (regulatory enzyme)  conserved as ATP.

o   Phosphate loss from PEP leads to pyruvate being formed in unstable enol form which spontaneously tautomerize into more stable keto form.

§  Second example of substrate level phosphorylation.

11.  Reduction of pyruvate to lactate.

o   Large NADH is produced in step 6  oxidized by reducing pyruvate to lactate by lactate dehydrogenase(LDH).

§  Final part of anaerobic glycolysis!!

§  The formation of lactate is the major fate for pyruvate in red blood cells (RBC), lens and cornea of the eye, kidney medulla, testes and leucocytes.

Fate of pyruvate under aerobic conditions

-       Pyruvate  acetyl-CoA (irreversible reaction in mitochondria by pyruvate dehydrogenase).

-       Acetyl-CoA enters Krebs cycle  metabolized to generate energy or substrate for synthesis of fatty acids.

Energy yielded in aerobic glycolysis.

Ø  2ATP molecules consumed in phase I.

Ø  4ATP molecules produced in phase II. (scroll up to first pic)

Energy yielded in anaerobic glycolysis.

Ø  1 glucose  4-2 = 2 ATP

Ø  Glucose + 2ADP + 2Pi  2 Lactate + 2ATP + 2H+.

Anaerobic release small amount of energy but valuable under conditions of low oxygen, during intensive exercise in muscles and in tissues with few mitochondria as in medulla of kidney, mature erythrocytes and leucocytes and during ischemia.

Regulation of glycolysis

-       By 3 enzymes catalysing irreversible reactions

o   Hexokinase

o   Phosphofructokinase

o   Pyruvate kinase

Phosphofructokinase

Ø  PFK is activated by fructose 2,6-bisphosphate only in the liver.

Ø  fructose 2,6-bisphosphate forms when high levels of F6P are present.

Ø  fructose 2,6-bisphosphate synthesized by PFK-2

Ø  PFK-2 is inhibited by phosphorylation of enzyme cAMP-dependent protein kinase.

Effect of hormones on PFK-2

2 activities:

-       Phosphokinase

-       Phosphatase

Pyruvate kinase

-       Liver: L-PK

o   Subjected to allosteric regulation and reversible phosphorylation.

o   In response to glucagon  L-PK is phosphorylated and inactivated so that liver stops consuming glucose when it is urgently needed by brain muscles.

-       Muscle cells: M-PK

o   Allosteric enzyme 

§  ADP is an allosteric activator.

§  ATP is an inhibitor.

What happens to pyruvate?

-       Pyruvate transported to mitochondria by pyruvate translocase.

-       Pyruvate dehydrogenase complex generates acetyl-CoA and enters Krebs cycle,

Pyruvate dehydrogenase complex

-       Coenzymes:

o   Thiamine (pyrophosphate thiamine (B1))

o   Lipoate (lipoic acid (antioxidant))

o   CoA (pantothenate (B5))

o   E-FAD (riboflavin (B2))

o   NAD+ (niacin (B3))

§  Three of the coenzymes of the complex are tightly bound to enzymes of the complex (TPP, lipoamide and FAD+).

§  two are employed as carriers of the products of PDH complex activity (CoA and NAD+).

Mechanism of action of PHD complex

1.     Pyruvate decarboxylase

a.     Thiamine pyrophosphate (TPP) removes COOH from pyruvate leaving 2 carbon fragment that binds to the TPP.

2.     Lipoamide reductase transacetylase

a.     Acetyl group is transferred to one lipoamide arm, and then to the other to position for CoA transfer.

3.     Dihydrolipoyl dehydrogenase

a.     acetyl group is transferred to CoASH; the reduced lipoamides transfer 2H to FAD  FADH2, and FADH2 passes H to NAD+  NADH

Net result:

-       Pyruvate + CoA + NAD+  C=2 + acetyl-CoA +NADH + H+ (irreversible reaction)

Krebs cycle

-       Pathway for oxidation of all fuel molecules including sugars, amino acids and fatty acids.

-       8 enzymes.

-       Degrades 2C atoms containing acetyl unit  2CO2.

-       Generates reduced coenzymes NADH and FADH2 and high energy compound GTP.

-       In mitochondrial matrix

-       Only in presence of oxygen

-       Provides intermediates for biosynthetic reactions.

1.     Condensation of acetyl-CoA with oxaloacetate.

o   Citrate synthase is an allosterically regulated enzyme

o   Enzyme removes a proton from CH3 on acetyl-CoA  Negatively charged CH2 forms carbonyl bond with oxaloacetate  Subsequent loss of CoA by hydrolysis drives reaction forward.

2.     Isomerisation of citrate to isocitrate.

o   Water is removed  water is added back and moves hydroxyl group from one carbon atom to its neighbour.

3.     Oxidative decarboxylation of isocitrate.

o   First of 4 oxidation steps the carbon containing the hydroxyl group  converted into carbonyl group intermediate product is unstable and losses CO2 (still

bound to enzyme).

§  Isocitrate dehydrogenase – allosterically regulated enzyme

4.     Oxidative decarboxylation of a-ketoglutarate.

o   a-ketoglutarate dehydrogenase complex resembles pyruvate dehydrogenase and catalyses oxidation NADH, CO2 and high energy thioester bond to CoA is formed.

o   Second oxidation step.

§  α-ketoglutarate dehydrogenase – similar to PDH. 5 coenzymes – TPP, NAD, FAD, CoA, lipoamide. Regulatory enzyme.

5.     Succinyl-CoA hydrolysis to succinate.

o   Phosphate molecule from solution displaces CoA  forming high energy phosphate linkage to succinate phosphate is passed to GDP  GTP

§  Succinyl-CoA – macroergic compound. Released energy is used to form GTP in substrate-level phosphorylation.

§  GTP+ADPATP +GDP

6.     Oxidation of succinate to fumarate.

o   Third step of oxidation  FAD removes 2 H+ atoms from succinate.

7.     Hydration of fumarate to malate.

o   Addition of water  places hydroxyl group next to carbonyl carbon.

8.     Regeneration of oxaloacetate.

o   Last oxidation step  carbon carrying hydroxyl group is converted to a carbonyl group  oxaloacetate is regenerated for step 1.

Regulation of Krebs cycle

3 enzymes:

-       Citrate synthetase.

o   Stimulated by ADP

o   Inhibited by ATP/NADH/citrate.

-       Isocitrate dehydrogenase.

o   Stimulated by ADP/Ca+2

o   Inhibited by ATP

-       a-Keto glutarate dehydrogenase.

o   Stimulated by Ca+2

o   Inhibited by Succ-CoA / NADH

Mitochondria

Ø  NADH and FADH2 produced in the Krebs cycle should immediately be re-oxidized to maintain Krebs cycle active.

Ø  Electrons from NADH and FADH2 can be passed to oxygen by a system of oxidoreductases called the electron transport chain.

Ø  Mitochondrion is the powerhouse of the cell.

Ø  System that couples respiration and ATP synthesis is called oxidative phosphorylation.

Ø  Oval shaped organelles with 2 membrane systems:

o   Outer membrane.

o   Highly folded inner membrane organized into layers (cristae)  increases surface area.

Ø  Membranes create two compartments:

o   Intermembranous space

o   Matrix.

Outer membrane

50% lipids, 50% proteins

-       Simple phospholipid bilayer containing proteins structures porins.

-       Porins render it permeable to molecules of 10 kilodaltons or less.

-       Ions, nutrients, ATP etc pass easily through.

Inner membrane

20% lipids, 80% proteins

-       Freely permeable only to oxygen and CO2.

-       Complex structure with all the complexes of electron transport chain, ATP synthetase complex and transport proteins.

Matrix

-       Contains enzymes responsible for citric acid cycle reactions and enzymes of fatty acid oxidation.

-       Contains dissolved oxygen, water, CO2, recyclable intermediates.

Electron transport chain

-       To liberate gradually energy from reduced coenzymes (NADH and FADH2) which are substrates of respiratory chain.

-       A set of proteins that interact with one another in pairs of redox reactions.

-       As high energy electrons move from one component of ETC to the next

o   First is oxidized and the second is reduced.

-       Drop of energy is used to make ATP.

Organization of the ETC

Four complexes:

-       I complex: NADH dehydrogenase.

-       II complex: succinate dehydrogenase

-       III complex: cytochromes bc1

-       IV complex: cytochromoxidase.

These are hydrophobic integral inner membrane proteins.

Electron carriers:

-       Coenzyme Q

-       Cytochrome C

Complex I

-       Protein part: 46 polypeptides

-       Cofactors:

o   FMN

o   22-24 iron-sulfur (fe-S) centers.

-       Responsible for transferring electrons from NADH to CoQ.

Complex II

-       Protein part: 4 polypeptides

-       Cofactors:

o   FAD

o   7-8 Fe-S

o   Cytochrome b560

-       Transfers electrons from succinate to CoQ.

CoQ – ubiquinone

-       Small mobile carrier, transferring electrons between complex I or II and cytochrome b

-       CoQ is restricted to the membrane phase because of its hydrophobic character. Like flavins, CoQ can undergo either 1- or 2-electron reactions leading to formation of the reduced quinol, the oxidized quinone, and the semiquinone intermediate.

-       Reduced CoQ diffuses in the lipid phase of the membrane and donates its electrons to complex III.

Complex III

-       Main components:

o   Heme proteins: cytochromes b and c1

o   Non-heme-iron protein: Rieske iron sulfur protein

-       Heme iron of all cytochromes participates in the cyclic redox reactions of the electron transport, alternating between oxidized Fe3+ and reduced Fe2+.

Cytochrome c

-       Smallest electron carrier of the cytochromes from complex III to IV.

Complex IV

-       Contains:

o   Hemoproteins: cytochrome a and cytochrome a3.

-       Copper containing proteins which oxidizes Cu+  Cu2+ during electron transfer through the complex to molecular oxygen.

Oxygen is final electron acceptor  water is final product of oxygen reduction.

Cause to electron flow and energy release.

Ø  Electron transfer down the ETC is exergonic due to difference in standard reduction potentials of electron carriers.

o   NAD+ reduction potential is -0,32V.

o   O2 reduction potential is +0,816V.

Ø  Therefore, electrons travel spontaneously from NAD to oxygen.

Electron transport and H+ release across inner membrane.

-       Matrix of inner membrane gains negative charge because of excess in OH- and is alkaline.

-       Intermembrane site gains positive charge and acidity because of excess in H+.

-       Difference in pH and charge is considered a proton motive force (electrochemical gradient).

-       ΔΨ is the difference between potentials on external and on internal surface of the Inner membrane of mitochondria.

-       ΔpH is the difference in pH on external and on internal surface of the inner membrane of mitochondria.

Proton motive force functions

1.     Electron potential

2.     Heat production.

3.     NADPH synthesis

4.     ATP

5.     Flagellar rotation

6.     Active transport.

ATP synthesis

-       As electrons passes through the complex’s protons are pumped into the intramembranous site.

-       Protons return to the matrix, down their concentration gradient by passing through ATP synthase coupling electron flow and proton pumping to ATP synthesis.

ATP synthase

-       A multiple subunit complex that binds ADP and inorganic phosphate at its catalytic site inside the mitochondrionand requires a proton gradient for activity in the forward direction.

o   F0: located in the inner membrane.

o   F1: protrudes from the inside of the inner membrane into the matrix.

Mechanism of ATP synthase

-       Proton channel permits protons to return into the matrix.

-       Proton flow results in conformational changes of catalytic subunits

-       Conformational changes benefit to ADP phosphorylation.

Conditions for oxidative phosphorylation:

1.     Inner membrane must be physically intact, so protons can only reenter by a process coupled to ATP synthesis.

2.     High proton concentration on the outside of the inner membrane.

Energy of the gradient is used to drive ATP synthesis as the protons travel down their thermodynamic gradient into the mitochondrion.

Chemiosmotic theory of oxidative phosphorylation

-       Coupling of ETC to ATP synthesis is indirect, via a H+ electrochemical gradient.

-       Respiration:

o   Spontaneous electron transfer through complexes I, III, and IV is coupled to non-spontaneous H+ ejection from the mitochondrial matrix. H+ ejection creates a membrane potential (negative in the matrix) and a pH gradient ( pH, alkaline in the matrix).

-       Phosphorylation by F1F0 ATP synthase:

o   Non-spontaneous ATP synthesis is coupled to spontaneous H+ transport into the matrix compartment. Respiration drives ATP synthesis indirectly, by creating the pH and electrical gradients that together are the driving force for H+ uptake. Return of protons to the matrix via Fo "uses up" the pH and electrical gradients.

Ø  ATP produced must exit to the cytosol to be used by transport pumps, kinases etc.

Ø  ADP and Pi arising from ATP hydrolysis must reenter mitochondria to convert into ATP.

Ø  Two carrier proteins are required for metabolic cycle.

o   Adenine Nucleotide Translocase (ADP/ATP carrier) is an antiporter that catalyzes exchange of ADP for ATP across the inner mitochondrial membrane. At cellular pH, ATP has four negative charges, while ADP has 3 negative charges. ADP3-/ATP4- exchange is thus driven by, and uses up, the membrane potential generated by respiration (one charge per ATP).

o   Phosphate reenters the mitochondrial matrix with H+, by an electroneutral symport mechanism. Pi entry is driven by and uses up the pH gradient (equivalent to one mole of H+ per mole of ATP).

Efficiency of oxidative phosphorylation

-       P/O ratio indicates:

o   How many inorganic phosphates are converted to ATP when 1 atom of O2 is consumed.

o   NADH P/O = 3

o   FADH2 P/O = 2

Regulation of oxidative phosphorylation

-       The flow of electrons through the electron transport system is regulated by the magnitude of the PMF (proton motive force).

o   The higher the PMF – the lower the rate of electron transport, and vice versa.

-       Resting conditions:

o   with a high cell energy charge, the demand for new synthesis of ATP is limited and, although the PMF is high, flow of protons back into the mitochondria through ATP synthase is minimal.

-       Increased energy demands:

o   Such as during vigorous muscle activity, cytosolic ADP rises and is exchanged with intramitochondrial ATP. Increased intramitochondrial concentrations of ADP cause the PMF to become discharged as protons pour through ATP synthase, regenerating the ATP pool. Thus, while the rate of electron transport is dependent on the PMF, the magnitude of the PMF at any moment simply reflects the energy charge of the cell.

Uncouplers & Ionophores

-       Small amphipathic molecules which dissolve in phospholipid bilayers and increase their ionic permeability.

o   Ionophores: transport a variety of ions.

o   Uncouplers: increase the proton permeability and disconnect the electron transport chain from the formation of ATP.

-       Protonophores increase the permeability of the inner mitochondrial membrane to H+.

o   CCCP: (carbonyl cyanide m-chloro phenyl hydrazone). This is a lipid-soluble weak acid which is a very powerful mitochondrial uncoupling agent.

Mechanism of uncoupling in brown adipose tissue

-       Some tissue mitochondria have specific proteins called uncoupling proteins.

-       When activated UCP-1 allows protons to flow across mitochondrial inner membrane, thus reducing electrochemical proton gradient and inhibiting ATP synthesis.

-       Energy released by electron flow through respiratory chain complexes is converted into heat in this case.

Reducing equivalent transport:

-       The inner mitochondrial membrane lacks an NADH transporter, and NADH produced in the cytosol cannot directly enter the mitochondrial matrix. However, reducing equivalents of NADH are transported from the cytosol into the matrix using substrate shuttles. In the glycerol 3- phosphate shuttle two electrons are transferred from NADH to dihydroxyacetone phosphate by cytosolic glycerol 3-phosphate dehydrogenase. The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme as FAD is reduced to FADH2. CoQ of the ETC oxidizes the FADH2.

-       Therefore, the glycerol 3-phosphate shuttle results in the synthesis of two ATP for each cytosolic NADH oxidized.

Malate-aspartate shuttle

-       The malate-aspartate shuttle produces NADH (rather than FADH2) in the mitochondrial matrix, thereby yieldingthree ATP for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate. A transport protein moves malate into the mitochondrial matrix.

Metabolism of glycogen

-       Constant source of blood glucose is an absolute requirement for life.

o   Main consumption in brain through aerobic pathway (75%).

o   Rest is used by skeletal muscle, erythrocytes, heart muscle.

-       Glucose comes from:

o   Diet

o   Gluconeogenesis

o   Glycogenolysis: Degradation of glycogen

-       Glucose is stored as glycogen.

o   Allows humans to eat intermittently by providing immediate source of blood glucose.

Glycogen

-       Stored in skeletal muscles and liver, other cells store minute amounts.

o   More glycogen in skeletal muscle because of greater mass

o   Concentration of glycogen higher in liver.

-       Fuel reserve for ATP synthesis during muscle contraction.

-       Liver glycogens maintain blood glucose concentration, particularly during early stages of fasting.

-       Stored in large granules of cytoplasm.

Liver glycogen

-       Stores increase during well fed stage.

-       Deplete during fast.

Muscle glycogen

-       Not affected by short period of fast

-       Muscle glycogen is synthesized to replenish muscle stores after they have been depleted,

-       Not available to other tissue because muscles lack glucose-6-phosphotase.

Glycogen structure

-       1 molecule: 10^8 daltons in molecular mass

-       Branched chain homopolysaccharide from a-D-glucose

-       Glucose residues linked by a-1,4 glycosidic bonds

-       After every 8-10 glycoyl residues a branch is created by a-1,6 glycosidic bonds.

Degradation of glycogen (glycogenolysis)

-       By the help of glycogen phosphorylase by removing single glucose residues from a-1,4-linkages.

-       Product = glucose-1-phosphate

-       This glucose is active (phosphorylated) which occurs without ATP hydrolysis.

Glycogen phosphorylase

-       Degrades glycogen chain until 4 glycosyl units remain on each chain before a branch point.

-       Resulting structure: limit dextrin

-       Cannot remove residues from a-1,6 linkages.

Removal of branch point

-       Done by debranching enzyme which have two activities:

o   Glucotransferase = oligo-(a-1,4a-1,4)-glucantransferase

§  Removes outer 3 of 4 glycosyl residues and transfer to the end of the chain.

o   Glucosidase = amylo-a-(1,6) glucosidase

§  Remaining single glucose residue is removed hydrolytically releasing free glucose.

-       The glycosyl chain is now available again for the degradation by glycogen phosphorylase until 4 glucosyl units from the next branch are reached.

Glucose-1-phosphate

-       Converted to glucose-6-phosphate by phosphoglucomutase.

-       Enzyme is present in liver and muscle.

Ø  Liver cells contain hydrolytic enzyme glucose-6-phosphotase that enables glucose to leave that organ. This occurs in liver, kidney and intestine but not in muscles.

o   In muscles glucose released will be oxidized in the glycolytic pathway because there are no glucose-6-phosphotase.

o   Activity of hexokinase in muscle is so high that any free glucose is immediately phosphorylated and enters glycolytic pathway.

Glycogen synthesis (glycogenesis)

-       Excess of glucose is used for synthesis of glycogen.

-       Occurs in cytosol.

-       Requires ATP for phosphorylation of glucose.

-       Done by glycogen synthase, uses two substrates: UDP and glycogen molecule.

1.     Glucose is phosphorylated

Glucose  hexokinase convert to G-6-P  phosphoglucomutase transfer phosphate to Carbon 1 G-1-P

-       Enzyme UDP-glucose pyrophosphorylase exchanges the phosphate on C-1 of glucose-1-phosphate for UDP.

-       Energy of phosphor-glycosyl bond of UDP-glucose is used by glycogen synthase to catalyse the incorporation of glucose into glycogen.

Primer

-       Fragment of glycogen serves as primer in cells whose glycogen stores are not totally depleted.

Totally depleted glycogen stores

-       Glycogenin:

o   first glycogen molecule acting as primer in glycogen synthesis when glycogen is absent.

o   Has intrinsic glycosyltransferase activity and catalyses transfer of glycosyl residues from UDP-glucose to tyrosine residue on glycogenin.

o   Catalyses transfer of 7 sequential glucosyl residues to the growing chain.

-       Glycogen synthase:

o   Comes after and ads further glucosyl residues with UDP-glucose.

Glycogen branches

-       Branching increases number of nonreducing ends to which new glucosyl residues can be added  increasing speed of reaction.

-       A-1,6-branvhes are produced by branching enzymes.

o   Enzyme transfers a terminal fragment of 6-7 glucose residues from at least 11 glucose residues.

o   Is further elongated by glycogen synthase.

Regulation of glycogen synthesis and degradation

Liver:

-       Accelerated synthesis during feeding.

-       Degradation during fasting.

Skeletal muscle:

-       Accumulation during muscle resting

-       Degradation during active exercise

2 levels:

I.               Glycogen synthase and glycogen phosphorylase are allosterically controlled.

a.     In the well fed state glycogen synthase is allosterically activated by G-6-P, when it is present in elevated concentrations.

b.     In contrast, glycogen phosphorylase is allosterically inhibited by G-6-P, as well as by ATP.

c.     Muscle glycogen phosphorylase is active in the presence of high AMP concentration, as occur in the muscle under extreme conditions of anoxia and ATP depletion.

II.             Reversible phosphorylation of glycogen phosphorylase and glycogen synthase.

a.     Glycogen synthase is active when not phosphorylated and inactive when phosphorylated.

b.     Glycogen phosphorylase is active when phosphorylated and inactive when non phosphorylated.

Hormone regulation:

-       Enzymes regulated by epinephrine, glucagone and insulin.

-       Cells of muscles and liver have specific receptors on the cell membrane and the binding of hormones to these receptors results in the formation of cAMP, that triggers all sequence of reactions.

cAMP pathway.

Ø  In response to lowered blood glucose cells of the pancreas secrete glucagon which binds to cell surface receptors on liver and several other cells.

Ø  In response to glucagon adenylate cyclase is activated.

Ø  Activation of adenylate cyclase leads to an increase in cAMP. cAMP binds to an enzyme called cAMP-dependent protein kinase, PKA

Ø  Active PKA phosphorylates the inactive form of phosphorylase kinase, resulting in its activation. Active phosphorylase kinase phosphorylates glycogen phosphorylase b, converting it into active phosphorylase a, which begins glycogen breakdown.

Ø  PKA also phosphorylates active form of glycogen synthase, resulting in its inactivation.

Ø  The identical cascade of events occurs in skeletal muscle cells.

Ø  However, in these cells the induction of the cascade is the result of epinephrine binding to receptors on the surface of muscle cells.

Ø  Epinephrine is released from the adrenal glands in response to neural signals indicating an immediate need for enhanced glucose utilization in muscle, the so-called fight or flight response.

Ø  Muscle cells lack glucagon receptors.

Gluconeogenesis – pentose phosphate pathway

-       Biosynthesis of new glucose from other metabolites.

-       Main function is to maintain constant blood glucose concentration.

-       Necessary for use as a fuel source by the brain, testes, erythrocytes, kidney medulla since glucose is the sole energy source for them.

-       Liver glycogen can only supply for 10-18 h and after that glucose must be formed from other metabolites during prolonged fasting.

-       Occurs 90% in the liver and 10% in the kidney.

Major precursors

-       Pyruvate

-       Lactate

-       Amino acids

-       Glycerol

Reactions

-       In this pathway pyruvate or lactate is converted into glucose.

-       Is not reversible of glycolysis.

-       7 reactions of glycolysis are reversible.

-       3 reactions (catalysed by hexokinase, phosphofructokinase, pyruvate kinase are irreversible).

o   Carboxylation of pyruvate  phosphoenolpyruvate

§  1: ATP requiring reaction by pyruvate carboxylase  oxaloacetate

§  PC contain biotin that is covalently bond to apoenzyme and can bind CO2.

§  2: PEP carboxykinase requires GTP in decarboxylation of OA to yield PEP.  PEP leaves mitochondria.

·      Oxaloacetate can convert to malate by malate dehydrogenase  Malate transferred to cytosol by malate/alpha-ketoglutarate transporter.

o   Decarboxylation and phosphorylation of cytosolic oxaloacetate.

§  In cytosol: malate dehydrogenase oxidize malate and PEPCK converts OA into PEP.

o   Dephosphorylation of F1,6-bisP.

§  Simple hydrolysis by fructose-1,6-bisphosphatase.

§  Major part of control of gluconeogenesis

o   Dephosphorylation of G-6-P.

§  Converts into glucose by glucose-6-phosphotase by hydrolysis.

§  Brain, muscle lack G-6-Pase and glucose is not used for blood glucose here.

Regulation

-       Negative effectors of glycolysis are positive effectors of gluconeogenesis.

-       Increase of AMP inhibits F-1,6-bisphosphotase  high ATP and low AMP stimulate gluconeogenesis

-       Allosteric regulation:

o   pyruvate carboxylase is activated by acetyl-CoA,

o   F-1,6-bisPase is stimulated by citrate

-       Regulation through amount of enzymes and synthesised after the need for glucose.

o   Amount increase in response to homones (F-1,6-biphosphatase, PEP carboxylase, glucose-6-phosphatase.  

Lactate:

-       Is released into blood by cells that lack mitochondria (blood cells and muscle)

-       Dead end of metabolism and must convert into pyruvate.

-       In liver lactate oxidize to pyruvate and convert into glucose by gluconeogenesis pathway.

-       Glucose is released to blood and taken up by skeletal muscle.  converted into lactate.

-       Cori cycle: relationship between glycolysis and glyconeogenesis.

Glycerol

-       Glycerol kinase convert to glycerol-3-phosphate and dehydrogenation to dihydroxyacetone phosphate by glyceraldehyde-3-phosphate dehydrogenase  enters glyconeogenesis and can convert into fructose-1,6-bisphosphate.

Amino acids

-       All except leucin, lysine can be degraded to Krebs cycle  carbon skeleton of the acids convert to OA and further to pyruvate.

-       During exertion and liver during fasting, catabolism of muscle proteins to amino acids contributes the major source of carbon for maintenance of blood glucose levels.

Pentose phosphate pathway

-       In order to carry on anabolic reactions, a cell need energy (ATP) and reducing power in form of NADPH.

-       NADPH is produced during glucose-6-P oxidation in this pathway.

-       Active in tissues involved in cholesterol and fatty acid synthesis (liver, adipose, adrenal cortex, mammary glands).

-       Low activity in heart and skeletal muscle.

Functions

-       Provides NADPH

-       Provides ribose 5-phosphate for synthesis of nucleotides and nucleic acids.

-       Converts glucose into other sugars.

-       In cells (particularly in erythrocytes), high levels of NADPH (produced by PPP) are necessary to maintain high reduction potential needed to prevent damaging effects of reactive oxygen species.

Enzymes are located in the cytoplasm.

2 stages:

1.     Irreversible oxidative branch:

a.     B glucose-6-phosphate and involves enzymes glucose-6- phosphate dehydrogenase, lactonase, 6-phosphogluconate dehydrogenase and phosphopentose isomerase. The product of this branch is D-ribose-5-phosphate.

2.     Reversible non-reducing branch.

a.     6 molecules of pentose phosphates are converted into 5 molecules of hexose phosphates.

-       First carbon of glucose-6-P is oxidized to a lactone.  2 electrons are released and reduce one molecule of NADP+  NADPH

-       Ring opens with reaction with water.

-       Next, decarboxylation of gluconate releases two more electrons, which reduce another NADP+ molecule. A five-carbon sugar, ribulose-5-phosphate, is produced in the reaction.  isomerize into ribose-5-P.

Non oxidative

-       6 molecules of pentose-5-P are converted into 4 molecules of fructose-6-P and 2 molecules of glyceraldehyde-3-P.

Ø  Fructose-6-P and glyceraldehyde-3-P can be degraded by glycolysis in order to produce energy, or recycled through gluconeogenesis to regenerate glucose-6-P.

Ø  When demand for ribose-5-P is larger than the NADPH demand  non-oxidative part can operate in reverse and yield 3 ribose-5-P and 2 F-6-P and 1 glyceraldehyde-3-P.

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Regulation

-       NADPH concentration

o   High supresses the rate-limiting glucose-6-P dehydrogenase reaction.

-       Glucose-6-P dehydrogenase is also regulated at expression level: its expression increases when diet contains large amounts of carbohydrates.

Fructose metabolism

-       Enters glycolysis as GAP and DAP by a series of reactions that occurs in the liver: action of fructokinase and aldolase.

-       In other tissues - action of hexokinase

Galactose metabolism

-       Converted into G-6-P through 5 steps: