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Chapter 20 Electron Transport and Oxidative Phosphorylation

Dr Khairul Ansari

20.1 Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur?
Electron Transport: Electrons carried by reduced coenzymes (NADH and [FADH2] are passed through a chain of proteins and coenzymes to drive the generation of a proton gradient across the inner mitochondrial membrane Oxidative Phosphorylation: The proton gradient drives the synthesis of ATP It all happens in or at the inner mitochondrial membrane

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20.1 Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur?

Figure 20.1 (a) A drawing of a mitochondrion. (b) Tomography of a rat liver mitochondrion. The colors represent individual cristae.

20.2 How Is the Electron Transport Chain Organized?

Four protein complexes in the inner mitochondrial membrane A lipid-soluble coenzyme (UQ, CoQ) and a water-soluble protein (cytochrome c) shuttle between protein complexes Electrons generally fall in energy through the chain - from complexes I and II to complex IV

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20.2 How Is the Electron Transport Chain Organized?

Figure 20.2 Reduction potentials for the components of the mitochondrial electrontransport chain. Values indicated are consensus values for animal mitochondria. Black bars represent o'; red bars, .

20.2 How Is the Electron Transport Chain Organized?

Figure 20.3 An overview of the complexes and pathways in the mitochondrial electron-transport chain.

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20.2 How Is the Electron Transport Chain Organized?

Complex I Oxidizes NADH and Reduces Coenzyme Q

NADH-CoQ Reductase/NADH dehydrogenase Complex I carries out electron transfer from NADH to Coenzyme Q The electron path: NADH FMN Fe-S UQ FeS UQ Four H+ transported out per 2 e-

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Complex I Transports Protons From the Matrix to the Cytosol

Figure 20.5 (a) Structural organization of mammalian Complex I, based on electron microscopy, showing functional relationships within the Lshaped complex. Electron flow from NADH to UQH2 in the membrane pool is indicated.

Complex I Transports Protons From the Matrix to the Cytosol

Figure 20.5 (b) Structure of the hydrophilic domain of Complex I from Thermus thermophilus is shown on a model of the membraneassociated complex.

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Complex I Transports Protons From the Matrix to the Cytosol

Figure 20.5 (c) Arrangement of the redox centers in Complex I. The various iron-sulfur centers of Complex I are designated by capital N.

Complex I Transports Protons From the Matrix to the Cytosol

Figure 20.5 (d) Blue arrows indicate the pathway of electron transfer from NADH to coenzyme Q. The energy of electron transfer allows a proton to cross Complex I near the interface between its hydrophilic and hydrophobic domains. Conformational changes accompanying these events push the long helical rod (magenta), reorienting residues in the associated subunits, so that protons bound there can cross into the intermembrane space.

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Solving a Medical Mystery Revolutionized Our Treatment of Parkinsons Disease

Cases of paralysis among illegal drug users in 1982 was traced to synthetic heroin that contained MPTP as a contaminant MPTP is converted rapidly in the brain to MPP+ MPP+ is a potent inhibitor of mitochondrial Complex I Such inhibition occurs especially in regions of the brain that deteriorate in Parkinsons disease Treatment of the paralysis victims with L-Dopa restored normal movement Implantation of fetal brain tissue also worked These treatments revolutionized the use of tissue implantation to treat neurodegenerative diseases

Complex II Oxidizes Succinate and Reduces Coenzyme Q

Succinate-CoQ Reductase/succinate dehydrogenase (only TCA cycle enzyme that is an integral membrane protein in the inner mitochondrial membrane) Also known as flavoprotein 2 (FP2) - FAD covalently bound four subunits, including 2 Fe-S proteins Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S, 2Fe-2S Path: succinate FADH2 2Fe2+ UQH2 Net reaction: succinate + UQ fumarate + UQH2

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Complex II Oxidizes Succinate and Reduces Coenzyme Q

Figure 20.6 A scheme for electron flow in Complex II. Oxidation of succinate occurs with reduction of [FAD]. Electrons are then passed to Fe-S centers and then to CoQ.

Fatty-Acyl-CoA Dehydrogenases Also Supply Electrons to UQ

Figure 20.7 The fatty acyl-CoA dehydrogenase reaction, emphasizing that the reaction involves reduction of enzyme-bound FAD (indicated by brackets).

The fatty acyl-CoA dehydrogenases are three soluble matrix enzymes involved in fatty acid oxidation (See also Chapter 23).

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Complex III Mediates Electron Transport from Coenzyme Q to Cytochrome c

UQ-Cytochrome c Reductase CoQ (UQ) passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle The principal transmembrane protein in complex III is the b cytochrome - with hemes bL and bH Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents UQH2 is a lipid-soluble electron carrier Cytochrome c is a water-soluble electron carrier

Complex III Mediates Electron Transport from Coenzyme Q to Cytochrome c

Figure 20.9 The structures of iron protoporphyrin IX, heme c, and heme a.

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Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side
Cytochrome c Oxidase Electrons from cytochrome c are used in a four-electron reduction of O2 to produce 2H2O Oxygen is thus the terminal acceptor of electrons in the electron transport pathway Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2 copper sites Complex IV also transports H+ across the inner mitochondrial membrane Four H+ participate in O2 reduction and four H+ are transported in each catalytic cycle

Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side
Figure 20.13 Bovine cytochrome c oxidase consists of 13 subunits. The 3 largest subunits I (purple), II (yellow), and III (blue) contain the proton channels and the redox centers.

Subunits I, II, and III are common to most organisms. This minimal complex is sufficient to carry out both oxygen reduction and proton transport.

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Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side
Figure 20.14 The complete structure of bovine cytochrome c oxidase. As in Figure 20.13, subunit I is purple, subunit II is yellow, and subunit III is blue.

Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side Figure 20.15 The electrontransfer pathway for cytochrome c oxidase. Cytochrome c binds on the cytosolic face, transferring electrons through the copper and heme centers to reduce O2 on the matrix side of the membrane.

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The Complexes of Electron Transport May Function as Supercomplexes

For many years, the complexes of the electron transport chain were thought to exist and function independently in the mitochondrial inner membrane However, growing experimental evidence supports the existence of multimeric supercomplexes of the four electron transport complexes Also known as respirasomes, these complexes may represent functional states Association of two or more of the individual complexes may be advantageous for the organism

A model for the electron-transport pathway in the mitochondrial inner membrane.

Figure 20.18 (b) A model for the electron-transport pathway in the mitochondrial inner membrane. UQ/UQH2 and cyt c are mobile carriers and transfer electrons between the complexes.

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Electron Transfer Energy Stored in a Proton Gradient: The Mitchell Hypothesis

The coupling between oxidation and phosphorylation was a mystery for many years Many biochemists squandered careers searching for the elusive "high energy intermediate" Peter Mitchell proposed a novel idea - a proton gradient across the inner membrane could be used to drive ATP synthesis The proton gradient is created by the proteins of the electrontransport pathway (Figure 20.19) Mitchell was ridiculed, but the chemiosmotic hypothesis eventually won him a Nobel prize Be able to calculate the G for a proton gradient (Equation 20.23)

Electron Transfer Energy Stored in a Proton Gradient: The Mitchell Hypothesis

Figure 20.19 The proton and electrochemical gradients existing across the inner mitochondrial membrane.

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20.3 What Are the Thermodynamic Implications of Chemiosmotic Coupling?

How much energy can be stored in an electrochemical gradient? The free energy difference for protons across the inner mitochondrial membrane includes a term for the concentration difference and a term for the electrical potential:

G = RT ln

[c2 ] + ZY [c1 ]

c1 and c2 are proton concentrations on the two sides of the membrane, Z is the proton charge, F is Faradays constant, and is the potential difference across the membrane

20.4 How Does a Proton Gradient Drive the Synthesis of ATP?

Proton diffusion through the ATP synthase drives ATP synthesis The ATP synthase consists of two parts: F1 and F0 (latter was originally named "Fo" for its inhibition by oligomycin) See Figure 20.20 and Table 20.2 for details F1 consists of five polypeptides: , ,, , and F0 includes three hydrophobic subunits denoted a, b and c F0 forms the transmembrane pore or channel through which protons move to drive ATP synthesis The a and b subunits comprise part of the stator and a ring of c subunits forms a rotor

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ATP Synthase is Composed of F1 and F0

Figure 20.20 The ATP synthase, a rotating molecular motor. The c, and subunits constitute the rotating portion (the rotor) of the motor. The b, d and h subunits form a long, slender stalk that connects F0 in the membrane and F1. Flow of protons from the asubunit through the c-subunits turns the rotor and drives the cycle of conformation changes in the - and -subunits of F1 that synthesize ATP.

ATP Synthase is Composed of F1 and F0

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The Catalytic Sites of ATP Synthase Adopt Three Different Conformations

Figure 20.21 (a) An axial view of the F1 unit of the ATP synthase; (b) A side view of the F1 unit with one and one subunit removed to show how the subunit (red) extends through the center of the hexamer.

John Walker Determined the Structure of the F1 Portion of ATP Synthase

In Walkers crystal structure of F1, one of the subunits contains AMP-PNP, one contains ADP, and the third site is empty the three states of Boyers model!

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Boyers 18O Exchange Experiment Identified the Energy Requiring Step

Figure 20.22 ATP-ADP exchange in the absence of a proton gradient. Exchange leads to incorporation of 18O in phosphate as shown. Boyers experiments showed that 18O could be incorporated into all four positions of phosphate, demonstrating that the free energy change for ATP formation from enzymebound ADP + Pi is close to zero.

Boyers Binding Change Mechanism Describes Events of Rotational Catalysis

Paul Boyer proposed that, at any instant: the three -subunits of F1 exist in three different conformations these different states represent the three steps of ATP synthesis each site steps through the three conformations or states to make ATP In Boyers binding change mechanism, the three catalytic sites thus cycle through the three intermediate states of ATP synthesis

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The Binding Change Mechanism

Figure 20.23 The binding change mechanism for ATP synthesis by ATP synthase. This model assumes that F1 has three interacting and conformationally distinct active sites: an open (O) conformation with almost no affinity for ligands, a loose (L) conformation with low affinity for ligands, and a tight (T) conformation with high affinity for ligands.

Proton Flow Through F0 Drives Rotation of the Motor and Synthesis of ATP

Figure 20.24 (a) Protons entering the inlet halfchannel in the -subunit are transferred to binding sites on c-subunits. Rotation of the c-ring delivers protons to the outlet half-channel in the subunit. Flow of protons through the structure turns the rotor and drives the cycle of conformational changes in that synthesize ATP.

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Racker and Stoeckenius Confirmed the Mitchell Model in a Reconstitution Experiment

Figure 20.25 The reconstituted vesicles containing ATP synthase and bacteriorhodopsin used by Stoeckenius and Racker to confirm the Mitchell chemiosmotic hypothesis. Upon illumination, bacteriorhodopsin pumped protons into these vesicles, and the resulting proton gradient was sufficient to drive ATP synthesis by the ATP synthase.

Inhibitors of Oxidative Phosphorylation Reveal Insights About the Mechanism

Many details of electron transport and oxidative phosphorylation have been learned from studying the effects of inhibitors Rotenone inhibits Complex I - and helps natives of the Amazon rain forest catch fish (Natives have learned to beat the roots of certain trees along river banks, releasing rotenone, which paralyzes the fish, making them easy prey) Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe3+) of a3 Oligomycin is an ATP synthase inhibitor

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Inhibitors of Oxidative Phosphorylation Reveal Insights About the Mechanism

Figure 20.27 The sites of action of several inhibitors of electron transport and oxidative phosphorylation.

Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthase

Uncoupling e- transport and oxidative phosphorylation Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient Uncouplers are hydrophobic molecules with a dissociable proton They shuttle back and forth across the membrane, carrying protons to dissipate the gradient

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Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthase

Figure 20.28 Structures of several uncouplers, molecules that dissipate the proton gradient across the inner mitochondrial membrane and thereby destroy the tight coupling between electron transport and the ATP synthase reaction.

Hibernating Animals Generate Heat by Uncoupling Oxidative Phosphorylation

Grizzly Bear

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Some Plants Use Uncoupled Proton Transport to Raise the Temperature of Floral Spikes

Skunk Cabbage

Hibernating Animals Generate Heat by Uncoupling Oxidative Phosphorylation

Chipmunk

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Some Plants Use Uncoupled Proton Transport to Raise the Temperature of Floral Spikes

Philodendron

ATP-ADP Translocase Mediates the Movement of ATP & ADP Across the Mitochondrial Membrane
ATP must be transported out of the mitochondria ATP out, ADP in - through a "translocase" ATP movement out is favored because the cytosol is "+" relative to the "-" matrix But ATP out and ADP in is net movement of a negative charge out - equivalent to a H+ going in So every ATP transported out costs one H+ One ATP synthesis costs about 3 H+ Thus, making and exporting 1 ATP = 4H+

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ATP-ADP Translocase Mediates the Movement of ATP & ADP Across the Mitochondrial Membrane

Figure 20.29 (a) The bovine ATP-ADP translocase.

ATP-ADP Translocase Mediates the Movement of ATP & ADP Across the Mitochondrial Membrane

Figure 20.29 (b) Outward transport of ATP (via the ATPADP translocase) is favored by the membrane electrochemical potential.

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20.5 - What Is the P/O Ratio for Mitochondrial Electron Transport and Oxidative Phosphorylation?
How many ATP can be made per electron pair sent through the chain? The e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen 8 H+ per turn of c8 F0 rotor 3 ATP 3.7 H+ flow back into matrix per ATP to cytosol 10/3.7 = 2.7 ATP for electrons entering as NADH For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen 6/3.7 = 1.6 ATP for electrons entering as succinate

20.6 How Are the Electrons of Cytosolic NADH Fed into Electron Transport?
Most NADH used in electron transport is cytosolic and NADH doesn't cross the inner mitochondrial membrane What to do? "Shuttle systems" effect electron movement without actually carrying NADH Glycerophosphate shuttle stores electrons in glycerol-3-P, which transfers electrons to FAD Malate-aspartate shuttle uses malate to carry electrons across the membrane

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The Glycerophosphate Shuttle Ensures Efficient Use of Cytosolic NADH

Figure 20.30 The glycerophosphate shuttle couples cytosolic oxidation of NADH with mitochondrial reduction of [FAD].

The Malate-Aspartate Shuttle is Reversible

Figure 20.31 The malate-aspartate shuttle.

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The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used
See Table 20.3 32.0 ATP per glucose if glycerol-3-P shuttle used 34.2 ATP per glucose if malate-Asp shuttle used In bacteria - no mitochondria - no extra H+ used to export ATP to cytosol. Assuming 10 c-subunits per F0 rotor (as in E. coli): 10 H+/NADH and 10 H+/3 ATP = 3 ATP/NADH 6 H+/succ and 10 H+/3ATP = ~ 1.8ATP/FADH2 (10 NADH + 2 [FADH2])/glucose 33.6 ATP

The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used

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20.7 How Do Mitochondria Mediate Apoptosis?

Mitochondria play a significant role in apoptosis, the programmed death of cells Mitochondria do this in part, by partitioning some of the apoptotic activator molecules, e.g., cytochrome c Oxidation of bound cardiolipins releases cytochrome c from the inner membrane Opening of pores in the outer membrane releases cytochrome c from the mitochondria Binding of cytochrome c to Apaf-1 in the cytosol leads to assembly of apoptosomes, thus triggering the events of apoptosis

20.7 How Do Mitochondria Mediate Apoptosis?

Figure 20.32 (a) Cytochrome c is anchored at the inner mitochondrial membrane by association with cardiolipin. The peroxidase activity of cytochrome c oxidizes a cardiolipin lipid chain, releasing cytochrome c from the membrane.

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20.7 How Do Mitochondria Mediate Apoptosis?

Figure 20.32 (b) The opening of pores in the outer membrane, induced by a variety of triggering agents, releases cytochrome c to the cytosol, where it initiates the events of apoptosis.

Figure 20.32 (b, top)

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Figure 20.32 (b bottom)

20.7 How Do Mitochondria Mediate Apoptosis?

Figure 20.33 Apaf-1 is a multidomain protein, consisting of an Nterminal CARD, a nucleotide-binding and oligomerization domain (NOD), and several WD40 domains.

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20.7 How Do Mitochondria Mediate Apoptosis?

Figure 20.33 Binding of cytochrome c to the WD40 domains and ATP hydrolysis unlocks Apaf-1 to form the semi-open conformation. Nucleotide exchange leads to oligomerization and apoptosome formation.

20.7 How Do Mitochondria Mediate Apoptosis?

Figure 20.33 A model of the apoptosome, a wheel-like structure with molecules of cytochrome c bound to the WD40 domains, which extend outward like spokes.

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Chapter 22 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway

Dr Khairul Ansari

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22.1 What Is Gluconeogenesis, and How Does It Operate?

Gluconeogenesis is the generation (genesis) of "new (neo) 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 Glycogen stores are at least partially depleted in strenuous exercise So the body must be able to make its own glucose To restore the amount stored in glycogen and to sustain normal activity

The Chemistry of Glucose Monitoring Devices

Individuals with diabetes must measure their serum glucose concentration, often several times a day Computerized automated devices have made this task much easier These devices rely on the oxidation of glucose to gluconic acid by glucose oxidase A colored dye produced in this reaction is directly proportional to the amount of glucose in the sample The patient typically applies a drop of blood to a plastic test strip that is then inserted into the meter

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The Gluconeogenesis Pathway

The Gluconeogenesis Pathway

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The Gluconeogenesis Pathway

The Substrates for Gluconeogenesis Include Pyruvate, Lactate, and Amino Acids
Pyruvate, lactate, glycerol, amino acids and all TCA 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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Features of Gluconeogenesis
Occurs mainly in liver and kidneys Not the mere reversal of glycolysis for 2 reasons: 1) The G of glycolysis is -74 kJ/mol If gluconeogenesis were just the reverse of glycolysis, its G would be positive Energetics must change to make gluconeogenesis favorable 2) Reciprocal regulation of glycolysis and gluconeogenesis: When glycolysis is active, gluconeogenesis is turned off, and when gluconeogenesis is proceeding, glycolysis is turned off.

Gluconeogenesis - Something Borrowed, Something New

Gluconeogenesis retains seven steps of glycolysis: Steps 2 and 4-9 Three steps are replaced: Pyruvate carboxylase and PEP carboxykinase replace the pyruvate kinase reaction of glycolysis Fructose-1,6-bisphosphatase replaces the phosphofructokinase reaction of glycolysis Glucose-6-phosphatase replaces the hexokinase reaction of glycolysis 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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Pyruvate Carboxylase is a Biotin-Dependent Enzyme

Pyruvate is converted to oxaloacetate The reaction requires ATP and bicarbonate as substrates This is a hint that biotin is required Biotin is covalently linked to an active-site lysine Acetyl-CoA is an allosteric activator The mechanism (Figure 22.3) is typical of biotin chemistry Regulation: when ATP or acetyl-CoA are high, pyruvate enters gluconeogenesis Note the "conversion problem" in mitochondria

Pyruvate Carboxylase is a Biotin-Dependent Enzyme

The pyruvate carboxylase reaction is the initiation of the gluconeogenesis pathway Carboxylations that use bicarbonate as the carbon source require biotin as a coenzyme

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Pyruvate carboxylase is a compartmentalized reaction.

Figure 22.4 Pyruvate is converted to oxaloacetate in the mitochondria. Because oxaloacetate cannot be transported across the mitochondrial membrane, it must be reduced to malate, which is then transported to the cytosol and oxidized back to oxaloacetate before gluconeogenesis can continue.

PEP Carboxykinase Uses GTP and a Decarboxylation to Drive PEP Synthesis

PEP carboxykinase catalyzes the conversion of oxaloacetate to PEP Lots of energy is needed to drive this reaction Energy is provided in 2 ways: Decarboxylation is a favorable reaction GTP is hydrolyzed The GTP used here is equivalent to an ATP

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PEP Carboxykinase Uses GTP and a Decarboxylation to Drive PEP Synthesis

The CO2 added to pyruvate in the pyruvate carboxylase reaction is removed in the PEP carboxykinase reaction. The use of GTP here (by mammals and several other organisms) is equivalent to the consumption of an ATP, due to the activity of the nucleoside diphosphate kinase (see Figure 19.2).

Fructose-1,6-bisphosphatase
Hydrolysis of F-1,6-bisPase to F-6-P Thermodynamically favorable the G in liver is -8.6 kJ/mol This is an allosterically regulated enzyme: citrate stimulates fructose-2,6-bisphosphate inhibits AMP inhibits The inhibition by AMP is enhanced by fructose-2,6bisphosphate

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Fructose-1,6-bisphosphatase

The fructose-1,6-bisphosphatase reaction The value of G in the liver is -8.6 kJ/mol

Glucose-6-Phosphatase Converts Glucose-6-P to Glucose in the Endoplasmic Reticulum

Presence of G-6-Pase in ER of liver and kidney cells makes gluconeogenesis possible Muscle and brain do not do gluconeogenesis G-6-P is hydrolyzed after uptake into the ER The glucose-6-phosphatase system includes the phosphatase itself and three transport proteins, T1, T2, and T3. T1 takes glucose-6-P into the ER, where it is hydrolyzed by the phosphatase T2 and T3 export glucose and Pi, respectively, to the cytosol Glucose is exported to the circulation by GLUT2

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Glucose-6-Phosphatase Converts Glucose-6-P to Glucose in the Endoplasmic Reticulum

Figure 22.5 Glucose-6-phosphatase is localized in the ER.

Coupling with hydrolysis of ATP and GTP drives gluconeogenesis

The net reaction of conversion of pyruvate to glucose in gluconeogenesis is: 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 6 H2O glucose + 4ADP + 2 GDP + 6Pi + 2 NAD+

Net energy

G = - 37.7 KJ/mol

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Gluconeogenesis Inhibitors and Other Diabetes Therapy Strategies

Diabetes is the inability to assimilate and metabolize blood glucose (Type I-unable to synthesize and secrete insulin and type II- produce sufficient insulin but the metabolic pathway that response to insulin are defective) Metformin improves sensitivity to insulin by stimulating glucose uptake by glucose transporters Gluconeogenesis inhibitors may be the next wave of diabetes therapy 3-Mercaptopicolinate and hydrazine inhibit PEP carboxykinase Chlorogenic acid inhibits transport activity by the glucose-6phosphatase system S-3483 does the same, but binds a thousand times more tightly to the transporter

Lactate Formed in Muscles is Recycled to Glucose in the Liver

How your liver helps you during exercise: Recall that vigorous exercise can lead to a buildup of lactate and NADH, due to oxygen shortage and the need for more glycolysis NADH can be reoxidized during the reduction of pyruvate to lactate Lactate is then returned to the liver, where it can be reoxidized to pyruvate by liver LDH Liver provides glucose to muscle for exercise and then reprocesses lactate into new glucose

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22.2 How Is Gluconeogenesis Regulated?

Reciprocal control with glycolysis When glycolysis is turned on, gluconeogenesis should be turned off When energy status of cell is high, glycolysis should be off and pyruvate, etc., should be used for synthesis and storage of glucose When energy status is low, glucose should be rapidly degraded to provide energy The regulated steps of glycolysis are the very steps that are regulated in the reverse direction!

Figure 22.8 The principal regulatory mechanisms in glycolysis and gluconeogenesis. Allosteric activators are indicated by plus signs and allosteric inhibitors by minus signs.

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22.2 How Is Gluconeogenesis Regulated?

Allosteric and Substrate-Level Control Glucose-6-phosphatase is under substrate-level control, not allosteric control The fate of pyruvate depends on acetyl-CoA Fructose-1,6-bisphosphatase is inhibited by AMP, activated by citrate - the reverse of glycolysis Fructose-2,6-bisphosphate is an allosteric inhibitor of fructose1,6-bisphosphatase

Fructose-2,6-bisphosphate is a powerful inhibitor of fructose1,6-bisphosphatase

22.3 How Are Glycogen and Starch Catabolized in Animals?

Obtaining glucose from storage (or diet) -Amylase is an endoglycosidase It cleaves dietary amylopectin or glycogen to maltose, maltotriose and other small oligosaccharides It is active on either side of a branch point, but activity is reduced near the branch points Debranching enzyme cleaves "limit dextrins" Note the 2 activities of the debranching enzyme It transfers trisaccharide groups And cleaves the remaining single glucose units from the main chain

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22.3 How Are Glycogen and Starch Catabolized in Animals?

Figure 22.11 (a) The sites of hydrolysis of starch by - and amylases are indicated.

Metabolism of Tissue Glycogen is Regulated

Digestive breakdown of starch is unregulated - nearly 100% of ingested food is absorbed and metabolized But tissue glycogen is an important energy reservoir - its breakdown is carefully controlled Glycogen consists of "granules" of high MW Glycogen phosphorylase cleaves glucose from the nonreducing ends of glycogen molecules This is a phosphorolysis, not a hydrolysis Metabolic advantage: product is a sugar-P - a potential glycolysis substrate

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Metabolism of Tissue Glycogen is Regulated

Figure 22.13 The glycogen phosphorylase reaction. Phosphate is the attacking nucleophile, so this reaction is a phosphorolysis.

22.4 How Is Glycogen Synthesized?

Glucose units are activated for transfer by formation of sugar nucleotides What are other examples of "activation"? acetyl-CoA, biotin, THF Luis Leloir showed in the 1950s that glycogen synthesis depends on sugar nucleotides UDP-glucose pyrophosphorylase catalyzes a phosphoanhydride exchange (Figure 22.14) driven by pyrophosphate hydrolysis

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22.4 How Is Glycogen Synthesized?

UDP-glucose is one of the sugar nucleotides. Discovered by Luis Leloir in the 1950s, they are activated forms of sugar.

The mechanism of the UDPglucose pyrophosphorylase reaction. Attack by a phosphate oxygen of glucose-1-P on the phosphorus of UTP is followed by departure of the pyrophosphate anion.

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Glycogen Synthase Catalyzes Formation of (14) Glycosidic Bonds in Glycogen

Forms (1 4) glycosidic bonds in glycogen The very large glycogen particle is built around a single protein, glycogenin, at the core The first glucose is linked to a tyrosine -OH on the protein Sugar units are then added by the action of glycogen synthase Glycogen synthase transfers glucosyl units from UDP-glucose to C-4 hydroxyl at a nonreducing end of a glycogen strand. Note the oxonium ion intermediate (Fig. 22.15)

Figure 22.15 The glycogen synthase reaction.

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Figure 22.16 Formation of glycogen branches by the branching enzyme. Six- or seven-residue segments of a growing glycogen chain are transferred to the C-6 hydroxyl group of a glucose residue on the same or a nearby chain.

Advanced Glycation End Products A Serious Complication of Diabetes

Sugars can react nonenzymatically with proteins The C-1 carbonyl groups of glucose form Schiff bases linkages with lysine side chains of proteins These Schiff base adducts undergo Amadori rearrangements and subsequent oxidations to form irreversible glycation end products (AGEs) AGEs are implicated in circulation, joint, and vision problems in diabetics Measurement of glycated hemoglobin is a better diagnostic yardstick for type-2 diabetes than serum glucose levels

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RAGE Receptor for Advanced Glycation End-Products

RAGE is a multifunctional protein of the innate immune system that plays pivotal roles in diabetes, chronic inflammation, neurodegenerative diseases, T-lymphocyte proliferation, and cancer It is a 1-TMS protein with 3 extracellular domains The first two extracellular domains contain a basic patch and a hydrophobic patch Binding of its ligands depends on interactions with these surfaces, particularly the large basic patch comprised of Arg and Lys side chains Binding of ligands induces dimerization of RAGE and triggers intracellular responses related to inflammation and tumorigenesis

22.5 How Is Glycogen Metabolism Controlled?

A highly regulated process, involving reciprocal control of glycogen phosphorylase and glycogen synthase GP allosterically activated by AMP and inhibited by ATP, glucose-6-P and caffeine GS is stimulated by glucose-6-P Both enzymes are regulated by covalent modification phosphorylation

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Glycogen Synthase is Regulated by Covalent Modification

Glycogen synthase exists in two distinct forms Active, dephosphorylated GS-I Less active, phosphorylated GS-D The phosphorylated form is allosterically activated by glucose-6-P At least 9 serine residues are phosphorylated Four different protein kinases are involved Dephosphorylation is carried out by phosphoprotein phosphatase1 (PP1) PP1 inactivates glycogen phosphorylase and activates glycogen synthase

Insulin Modulates the Action of Glycogen Synthase in Several Ways

Binding of insulin to plasma membrane receptors in the liver and muscles triggers protein kinase cascades that stimulate glycogen synthesis Insulins effect include stimulation of lipid synthesis, glycogen synthesis, protein synthesis, glycolysis, and active transport, and inhibition of gluconeogenesis and lipid breakdown Glucose uptake provides substrate for glycogen synthesis and glucose-6-P, which allosterically activates the otherwise inactive form of glycogen synthase

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Insulin Modulates the Action of Glycogen Synthase in Several Ways

Figure 22.17 Insulin triggers protein kinases that stimulate glycogen synthesis.

The Actions of Insulin on Metabolism

Figure 22.17b The metabolic effects of insulin are mediated through protein phosphorylation and second messenger modulation.

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Hormones Regulate Glycogen Synthesis and Degradation

Storage and utilization of tissue glycogen and other aspects of metabolism are regulated by hormones, including glucagon, epinephrine, and the glucocorticoids Insulin is a response to increased blood glucose Insulin triggers glycogen synthesis when blood glucose rises Between meals, blood glucose is 70-90 mg/dL Glucose rises to 150 m/dL after a meal and then returns to normal within 2-3 hours Glucagon and epinephrine stimulate glycogen breakdown

Hormones Regulate Glycogen Synthesis and Degradation

Insulin is secreted from the pancreas (to liver) in response to an increase in blood glucose Note that the portal vein is the only vein in the body that feeds an organ Insulin acts to lower blood glucose rapidly in several ways, stimulating glycogen synthesis and inhibiting glycogen breakdown

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Hormones Regulate Glycogen Synthesis and Degradation

Figure 22.18 The portal vein system carries pancreatic secretions such as insulin and glucagon to the liver.

Glucagon and Epinephrine Stimulate Glycogen Breakdown and Inhibit Glycogen Synthesis
Glucagon and epinephrine stimulate glycogen breakdown the opposite effect of insulin Glucagon (a 29-residue peptide) is also secreted by pancreas Glucagon acts in liver and adipose tissue only Epinephrine (adrenaline) is released from adrenal glands Epinephrine acts on liver and muscles When either hormone binds to its receptor on the outside surface of the cell membrane, a phosphorylase cascade amplifies the signal

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Figure 22.20 Glucagon and epinephrine activate a cascade of reactions that stimulate glycogen breakdown and inhibit glycogen synthesis in liver and muscles, respectively.

Glucagon and Epinephrine Actions in Liver and Muscle

Figure 22.20 Glucagon and epinephrine each activate glycogen breakdown and inhibit glycogen synthesis in liver and muscles, respectively. In both tissues, binding of glucagon or epinephrine activates adenylyl cyclase, initiating the cascade.

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Glucagon and Epinephrine Actions in Liver and Muscle

Figure 22.20. In liver, glucagon inhibits glycolysis and stimulates gluconeogenesis. In muscles, epinephrine stimulates glycolysis to provide energy for contraction.

Epinephrine and Glucagon

The difference: Both are glycogenolytic in liver, but for different reasons Epinephrine is the fight-or-flight hormone It rapidly mobilizes large amounts of energy Glucagon is for long-term maintenance of steady-state levels of glucose in the blood It activates glycogen breakdown It activates liver gluconeogenesis

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Cortisol and Glucocorticoids Exert a Variety of Effects on Glycogen Metabolism

Glucocorticoids are steroid hormones that exert distinct effects on liver, skeletal muscle, and adipose tissue Cortisol is primarily catabolic it promotes protein breakdown and decreases protein synthesis in skeletal muscle In liver, however, it stimulates gluconeogenesis and increases glycogen synthesis, by Stimulating expression of genes for gluconeogenic enzymes Activating enzymes of amino acid metabolism Stimulating the urea cycle (see Chapter 25)

Cortisol and Glucocorticoids Exert a Variety of Effects on Glycogen Metabolism

Figure 22.21 The effects of cortisol on carbohydrate and protein metabolism in the liver.

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22.6 Can Glucose Provide Electrons for Biosynthesis?

Cells are provided with a constant supply of NADPH for biosynthesis by the pentose phosphate pathway Also called the hexose monophosphate shunt This pathway also produces ribose-5-P This pathway consists of two oxidative processes followed by five non-oxidative steps It operates mostly in the cytosol of liver and adipose cells NADPH is used in cytosol for fatty acid synthesis

22.6 Can Glucose Provide Electrons for Biosynthesis?

Figure 22.22 The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.

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22.6 Can Glucose Provide Electrons for Biosynthesis?

Figure 22.22 The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.

Figure 22.22 The pentose phosphate pathway.

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The Oxidative Steps of the Pentose Phosphate Pathway

Glucose-6-P Dehydrogenase Irreversible 1st step - highly regulated! Gluconolactonase The uncatalyzed reaction happens, too 6-Phosphogluconate Dehydrogenase An oxidative decarboxylation (in that order)

The Oxidative Steps of the Pentose Phosphate Pathway

Figure 22.23 The glucose-6-phosphate dehydrogenase reaction is the committed step in the pentose phosphate pathway.

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Figure 22.24 The Gluconolactonase Reaction

Figure 22.24 The gluconolactonase reaction. The uncatalyzed reaction also occurs.

The Oxidative Steps of the Pentose Phosphate Pathway

Figure 22.25 The 6-phosphogluconate dehydrogenase reaction. Initial NADP+-dependent dehydrogenation yields a -keto acid, 3keto-6-phosphogluconate, which is very susceptible to decarboxyation (the second step). The resulting product, D-ribulose5-P, is the substrate for the nonoxidative reactions of the pentose phosphate pathway.

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The Nonoxidative Steps of the Pentose Phosphate Pathway

Five steps, but only 4 types of reaction... Phosphopentose isomerase converts ketose to aldose Phosphopentose epimerase epimerizes at C-3 Transketolase ( a TPP-dependent reaction) transfer of two-carbon units Transaldolase (uses a Schiff base mechanism) transfers a three-carbon unit

The Nonoxidative Steps of the Pentose Phosphate Pathway

Figure 22.26 The phosphopentose isomerase reaction converts a ketose to an aldose. The reaction involves an enediol intermediate.

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The Nonoxidative Steps of the Pentose Phosphate Pathway

Figure 22.27 The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate. The mechanism involves an enediol intermediate and occurs with inversion at C-3.

The Nonoxidative Steps of the Pentose Phosphate Pathway

Figure 22.28 The transketolase reaction of step 6 in the pentose phosphate pathway. This is a two-carbon transfer reaction that requires thiamine pyrophosphate as a coenzyme. TPP chemistry was discussed in Chapter 19 (see the pyruvate dehydrogenase reaction).

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The Nonoxidative Steps of the Pentose Phosphate Pathway

Figure 22.29 The transketolase reaction of step 8 in the pentose phosphate pathway. This is another two-carbon transfer, and it also requires TPP as a coenzyme.

The Nonoxidative Steps of the Pentose Phosphate Pathway

Figure 22.30 The mechanism of the TPP-dependent transketolase reaction. The group transferred is really an aldol. Despite this, the name transketolase persists.

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Figure 22.30 The mechanism of the TPP-dependent transketolase reaction. The group transferred is really an aldol. Despite this, the name transketolase persists.

The Nonoxidative Steps of the Pentose Phosphate Pathway

Figure 22.31 The transaldolase reaction. In this reaction, a 3carbon unit is transferred, first to an active-site lysine, and then to the acceptor molecule.

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Figure 22.32 The transaldolase mechanism involves attack on the substrate by an active-site lysine. Departure of erythrose-4-P leaves the reactive enamine, which attacks the aldehyde carbon of Gly-3-P.

Utilization of Glucose-6-P Depends on the Cells Need for ATP, NADPH, and Rib-5-P
Glucose can be a substrate either for glycolysis or for the pentose phosphate pathway The choice depends on the relative needs of the cell for biosynthesis and for energy from metabolism ATP can be made if G-6-P is sent to glycolysis Or, if NADPH or ribose-5-P are needed for biosynthesis, G-6-P can be directed to the pentose phosphate pathway Depending on these relative needs, the reactions of glycolysis and the pentose phosphate pathway can be combined in four principal ways

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Four Ways to Combine the Reactions of Glycolysis and Pentose Phosphate

1) Both ribose-5-P and NADPH are needed by the cell In this case, the first four reactions of the pentose phosphate pathway predominate NADPH is produced and ribose-5-P is the principal product of carbon metabolism 2) More ribose-5-P than NADPH is needed by the cell Synthesis of ribose-5-P can be accomplished without making NADPH, by bypassing the oxidative reactions of the pentose phosphate pathway

Four Ways to Combine the Reactions of Glycolysis and Pentose Phosphate

Case 1: Both ribose-5-P and NADPH are needed

Figure 22.33 When biosynthetic demands dictate, the first four reactions of the pentose phosphate pathway predominate and the principal products are ribose-5-P and NADPH.

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Four Ways to Combine the Reactions of Glycolysis and Pentose Phosphate

3) More NADPH than ribose-5-P is needed by the cell This can be accomplished if ribose-5-P produced in the pentose phosphate pathway is redirected to produce glycolytic intermediates 4) Both NADPH and ATP are needed by the cell, but ribose-5-P is not This can be done by redirecting ribose-5-P, as in case 3 above, if fructose-6-P and glyceraldehyde-3P made in this way proceed through glycolysis to produce ATP and pyruvate, and pyruvate continues through the TCA cycle to make more ATP

Integrating the Warburg Effect ATP consumption promotes cancer metabolism Cancer cells use glycolytic intermediates for the synthesis of macromolecules and must balance their ATP requirements and biosynthetic needs Xiaodong Wang has shown that EMTPD5, a UDPase in the ER is associated with ATP consumption in tumor cells UMP made in this reaction is transported into the cytosol, where it is converted back to UDP, then UTP, and then to UDP-glucose Ensuing reactions send glycolytic intermediates to the pentose phosphate pathway, helping cancer cells to fine-tune production of NADPH, ribose-5-P, and ATP

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Cellular Signals Modulate Production of NADPH, ribose5-P, and ATP

ENTPD5 expression is enhanced in tumors and promotes ATP consumption, stimulating aerobic glycolysis. PI3K and AKT stimulate glucose uptake, as well as hexokinase and PFK. AMP kinase inhibits PFK, whereas p53 blocks synthesis of NADPH by inhibition of G6P dehydrogenase.

Xylulose-5-Phosphate is a Metabolic Regulator

In addition to its role in the pentose phosphate pathway, xylulose-5-P is also a signaling molecule When blood glucose rises, glycolysis and the pentose phosphate pathways are activated in the liver The latter pathway makes xylulose-5-P, which stimulates protein phosphatase 2A (PP2A) PP2A dephosphorylates PFK-2/F-2,6-BPase and also carbohydrate-responsive element-binding protein (ChREBP) Glycolysis and lipid biosynthesis are both activated as a result

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Xylulose-5-Phosphate is a Metabolic Regulator

Activation of PP2A triggers dephosphorylation of PFK-2/F2,6-BPase, which raises F-2,6-BP levels, activating glycolysis and inhibiting gluconeogenesis.

Figure 22.34

Dephosphorylation of ChREBP elevates expression of genes for lipogenesis.

Aldose Reductase and Diabetic Cataract Formation

The complications of diabetes include a high propensity for cataract formation in later life Hyperglycemia is the cause, but why? Evidence points to the polyol pathway, in which glucose and other simple sugars are reduced in NADPH-dependent reactions Glucose is reduced by aldose reductase to sorbitol, which accumulates in lens fiber cells, increasing pressure and eventually rupturing the cells Aldose reductase inhibitors such as tolrestat and epalrestat suppress cataract formation

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Aldose Reductase and Diabetic Cataract Formation

Glucose is reduced by aldose reductase to sorbitol, which accumulates in lens fiber cells, increasing pressure and eventually rupturing the cells Aldose reductase inhibitors such as tolrestat and epalrestat suppress cataract formation

Aldose Reductase and Diabetic Cataract Formation

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