chem 4311- chapter20& 22

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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 (NADHand [FADH2] are passed through a chain of proteins and coenzymes todrive the generation of a proton gradient across the innermitochondrial membrane

Oxidative Phosphorylation: The proton gradient drives the synthesisof 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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Figure 20.2 Reduction

potentials for the

components of the

mitochondrial electron-

transport chain. Values

indicated are consensus

values for animal

mitochondria. Black bars

represento'; red bars, .


Figure 20.3 An overview of the complexes and pathways in the

mitochondrial electron-transport chain.

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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 L-

shaped complex. Electron flow

from NADH to UQH2 in themembrane pool is indicated.


Figure 20.5 (b) Structure of

the hydrophilic domain of

Complex I from Thermusthermophilus is shown on a

model of the membrane-

associated complex.

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Figure 20.5 (c) Arrangement of the

redox centers in Complex I. The

various iron-sulfur centers of

Complex I are designated by capitalN.


Figure 20.5 (d) Blue arrows indicate the pathway of electron transfer

from NADH to coenzyme Q. The energy of electron transfer allows aproton 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 tosynthetic 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 thatdeteriorate in Parkinsons disease

Treatment of the paralysis victims with L-Dopa restored normalmovement

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 TCAcycle enzyme that is an integral membrane protein in the innermitochondrial 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 ofsuccinate 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 bcytochrome - with hemes bL and bH

Cytochromes, like Fe in Fe-S clusters, are one- electron transferagents

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 cto

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 electrontransport pathway

Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2 coppersites

Complex IV also transports H+ across the inner mitochondrialmembrane

Four H+ participate in O2 reduction and four H+ are transported in

each catalytic cycle



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 outboth oxygen reduction and

proton transport.

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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 cto Reduce

Oxygen on the Matrix Side

Figure 20.15 The electron-

transfer 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 electrontransport chain were thought to exist and functionindependently in the mitochondrial inner membrane

However, growing experimental evidence supports theexistence of multimeric supercomplexes of the fourelectron transport complexes

Also known as respirasomes, these complexes mayrepresent functional states

Association of two or more of the individual complexesmay 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 mysteryfor many years

Many biochemists squandered careers searching for the elusive"high energy intermediate"

Peter Mitchell proposed a novel idea - a proton gradient across theinner membrane could be used to drive ATP synthesis

The proton gradient is created by the proteins of the electron-transport pathway (Figure 20.19)

Mitchell was ridiculed, but the chemiosmotic hypothesis eventuallywon 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:

c1 and c2 are proton concentrations on the two sides of themembrane, Z is the proton charge, F is Faradays constant,

and is the potential difference across the membrane

G =RTln[c

2]

[c1]+ZY

20.4 How Does a Proton Gradient Drive the Synthesis of

ATP?

Proton diffusion through theATP synthase drives ATP synthesis

The ATP synthase consists of two parts: F1 and F0 (latter wasoriginally 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 whichprotons move to drive ATP synthesis

The a and b subunits comprise part of the stator and a ring ofcsubunits 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 a-

subunit 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 theF1 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 of18O 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 enzyme-bound 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 half-

channel 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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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 theATP synthase.

Racker and Stoeckenius Confirmed the Mitchell Model in a

Reconstitution Experiment

Inhibitors of Oxidative Phosphorylation Reveal Insights About the

Mechanism

Many details of electron transport and oxidative phosphorylationhave been learned from studying the effects of inhibitors

Rotenone inhibits Complex I - and helps natives of the Amazon rainforest catch fish

(Natives have learned to beat the roots of certain trees along riverbanks, releasing rotenone, which paralyzes the fish, making themeasy prey)

Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferricform (Fe3+) ofa3

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 ofseveral 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 transportand oxidative phosphorylation by dissipating the proton gradient

Uncouplers are hydrophobic molecules with a dissociable proton

They shuttle back and forth across the membrane, carryingprotons 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 "+" relativeto the "-" matrix

But ATP out and ADP in is net movement of a negative chargeout - 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 ATP-

ADP 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 pairfrom 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 NADHdoesn't cross the inner mitochondrial membrane

What to do?

"Shuttle systems" effect electron movement without actuallycarrying NADH

Glycerophosphate shuttle stores electrons in glycerol-3-P,which transfers electrons to FAD

Malate-aspartate shuttle uses malate to carry electrons acrossthe 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 Themalate-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 programmeddeath 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


Figure 20.32 (a) Cytochrome c is anchored at the inner mitochondrial

membrane by association with cardiolipin. The peroxidase activity ofcytochrome c oxidizes a cardiolipin lipid chain, releasing cytochrome

c from the membrane.

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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)


Figure 20.33 Apaf-1 is a multidomain protein, consisting of an N-

terminal CARD, a nucleotide-binding and oligomerization domain

(NOD), and several WD40 domains.

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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.


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 normalactivity

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


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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 Gwould be positive

Energetics must change to make gluconeogenesis favorable

2) Reciprocal regulation of glycolysis and gluconeogenesis:

When glycolysis is active, gluconeogenesis is turned off, andwhen 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 pyruvatekinase reaction of glycolysis

Fructose-1,6-bisphosphatase replaces the phosphofructokinasereaction of glycolysis

Glucose-6-phosphatase replaces the hexokinase reaction ofglycolysis

The new reactions provide for a spontaneous pathway (G negativein 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 entersgluconeogenesis

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-2,6-

bisphosphate

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Fructose-1,6-bisphosphatase

The fructose-1,6-bisphosphatase reaction

The value ofG 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 ofliver and kidney cells makesgluconeogenesis 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 itselfand three transport proteins, T1, T2, and T3.

T1 takes glucose-6-P into the ER, where it is hydrolyzed by thephosphatase

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- producesufficient 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-6-

phosphatase 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 andNADH, 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 topyruvate by liver LDH

Liver provides glucose to muscle for exercise and then reprocesseslactate 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 andpyruvate, etc., should be used for synthesis and storage of glucose

When energy status is low, glucose should be rapidly degraded toprovide energy

The regulated steps of glycolysis are the very steps that areregulated 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, notallosteric control

The fate of pyruvate depends on acetyl-CoA

Fructose-1,6-bisphosphatase is inhibited by AMP, activated bycitrate - the reverse of glycolysis

Fructose-2,6-bisphosphate is an allosteric inhibitor of fructose-1,6-bisphosphatase

Fructose-2,6-bisphosphate is apowerful inhibitor of fructose-

1,6-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, maltotrioseand other small oligosaccharides

It is active on either side of a branch point, but activity is reducednear 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 mainchain

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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 ingestedfood is absorbed and metabolized

But tissue glycogen is an important energy reservoir - its breakdownis carefully controlled

Glycogen consists of "granules" of high MW

Glycogen phosphorylase cleaves glucose from the nonreducing endsof glycogen molecules

This is a phosphorolysis, not a hydrolysis

Metabolic advantage: product is a sugar-P - a potential glycolysissubstrate

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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 sugarnucleotides

What are other examples of "activation"?

acetyl-CoA, biotin, THF

Luis Leloir showed in the 1950s that glycogen synthesis dependson sugar nucleotides

UDP-glucose pyrophosphorylase catalyzes a phosphoanhydrideexchange (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 UDP-

glucose 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 (14) 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 toC-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 phosphatase-1 (PP1)

PP1 inactivates glycogen phosphorylase and activates glycogensynthase

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 ofmetabolism 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 tonormal within 2-3 hours

Glucagon and epinephrine stimulate glycogen breakdown


Insulin is secreted from the pancreas (to liver) in response toan increase in blood glucose

Note that the portal vein is the only vein in the body thatfeeds an organ

Insulin acts to lower blood glucose rapidly in several ways,stimulating glycogen synthesis and inhibiting glycogenbreakdown

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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 theopposite 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 surfaceof 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 ofglucose 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 biosynthesisby 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 fivenon-oxidative steps

It operates mostly in the cytosol of liver and adipose cells

NADPH is used in cytosol for fatty acid synthesis


Figure 22.22 The pentose

phosphate pathway. The

numerals in the blue circles

indicate the steps discussed in

the text.

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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)


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.


Figure 22.25 The 6-phosphogluconate dehydrogenase reaction.Initial NADP+-dependent dehydrogenation yields a -keto acid, 3-

keto-6-phosphogluconate, which is very susceptible to

decarboxyation (the second step). The resulting product, D-ribulose-

5-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


Figure 22.26 The phosphopentose isomerase reaction converts aketose to an aldose. The reaction involves an enediol

intermediate.

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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.


Figure 22.28 The transketolase reaction of step 6 in the pentosephosphate 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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Figure 22.29 The transketolase reaction of step 8 in the pentose

phosphate pathway. This is another two-carbon transfer, and italso requires TPP as a coenzyme.


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 Themechanism of the

TPP-dependent

transketolase

reaction. The group

transferred is really

an aldol. Despite

this, the name

transketolase

persists.


Figure 22.31 The transaldolase reaction. In this reaction, a 3-

carbon 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 ofGly-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 forthe pentose phosphate pathway

The choice depends on the relative needs of the cell forbiosynthesis 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 phosphatepathway

Depending on these relative needs, the reactions ofglycolysis and the pentose phosphate pathway can becombined 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 pentosephosphate pathway predominate

NADPH is produced and ribose-5-P is the principalproduct of carbon metabolism

2) More ribose-5-P than NADPH is needed by the cell

Synthesis of ribose-5-P can be accomplished withoutmaking 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 toproduce glycolytic intermediates

4) Both NADPH and ATP are needed by the cell, butribose-5-P is not

This can be done by redirecting ribose-5-P, as incase 3 above, if fructose-6-P and glyceraldehyde-3-

P made in this way proceed through glycolysis toproduce ATP and pyruvate, and pyruvate continuesthrough 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 theirATP requirements and biosynthetic needs

Xiaodong Wang has shown that EMTPD5, a UDPase inthe ER is associated with ATP consumption in tumorcells

UMP made in this reaction is transported into thecytosol, where it is converted back to UDP, then UTP,and then to UDP-glucose

Ensuing reactions send glycolytic intermediates to thepentose phosphate pathway, helping cancer cells tofine-tune production of NADPH, ribose-5-P, and ATP

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Cellular Signals Modulate Production of NADPH, ribose-

5-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 phosphatepathway, xylulose-5-P is also a signaling molecule

When blood glucose rises, glycolysis and the pentosephosphate pathways are activated in the liver

The latter pathway makes xylulose-5-P, whichstimulates protein phosphatase 2A (PP2A)

PP2A dephosphorylates PFK-2/F-2,6-BPase and alsocarbohydrate-responsive element-binding protein

(ChREBP)

Glycolysis and lipid biosynthesis are both activated as aresult

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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.

Xylulose-5-Phosphate is a Metabolic Regulator

Aldose Reductase and Diabetic Cataract Formation

The complications of diabetes include a highpropensity for cataract formation in later life

Hyperglycemia is the cause, but why?

Evidence points to the polyol pathway, in whichglucose and other simple sugars are reduced inNADPH-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 andepalrestat suppress cataract formation

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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


chem 4311- chapter20& 22

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