CHAPTER 13
BIOCHEMICAL SIGNALLING
In general, every signaling pathway consists of a receptor protein that specifically binds a hormone or other ligand, a mechanism for
transmitting the ligand- binding event to the cell interior, and a series of intracellular responses that may involve the synthesis of a
second messenger and/or chemical changes catalyzed by kinases and phosphatases. These pathways often involve enzyme
cascades, in which a succession of events amplifies the signal. Three major pathways whereby intercellular signals are converted
(transduced) to intracellular signals:
(1) those that involve receptor tyrosine kinases
(2) utilize heterotrimeric G proteins
(3) employ phosphoinositide cascades
OVERVIEW OF CELL SIGNALLING
•
•
•
Cell signaling is part of a complex system of communication that governs basic cellular activities and coordinates
cell actions.
Cell-to-cell communication is essential for multicellular organisms.
The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue
repair, and immunity as well as normal tissue homeostasis.
A. Signaling Molecules
i. Signaling molecules could be lipids, phospholipids, amino acids, monoamines, proteins,
glycoproteins, or gases.
ii. Extracellular signal molecules can act over either short or long distances.
B. Signal Molecules Bind to Specific Receptors
i. Regardless of the nature of the signal, the target cell responds by means of a receptor protein, which
specifically binds the signal molecule and initiates a response.
ii. Signaling molecules binding surface receptors are generally large and hydrophilic, while those
entering the cell are generally small and hydrophobic.
C. Transduction Usually Involves Multiple Steps
i. Transduction: Cascades of molecular interactions relay signals from receptors to target molecules in
the cell
ii. At each step, the signal is transduced into a different form, usually a conformational change.
1. Multistep pathways can amplify a signal: A few molecules can produce a large cellular response.
2. Multistep pathways provide more opportunities for coordination and regulation
D. Many intracellular signaling proteins function as switches that are activated by phosphorylation
i. Signal is transmitted by a cascade of protein phosphorylation
E. Small Molecules and Ions as Second Messengers
i. Second messengers are small, nonprotein, water-soluble molecules or ions, such as cAMP, Ca2+,IP3
and DAG.
1. The extracellular signal molecule that binds to the membrane is a pathways “first messenger.”
2. Second messengers can readily spread throughout cells by diffusion.
3. Second messengers participate in pathways initiated by G-protein-linked receptors and receptor
tyrosine kinases.
F. Cyclic AMP
i. Cyclic AMP (cAMP) is one of the most widely used second messengers.
1. Adenylyl cyclase, an enzyme in the plasma membrane, converts ATP to cAMP in response to an
extracellular signal.
2. Cyclic AMP dependent protein kinase A (PKA) mediates most of the effects of cyclic AMP
ii.
iii.
Second Messenger cAMP in G Protein Signaling
Pathway
Response: Cell signaling leads to regulation of
transcription or cytoplasmic activities
1. HORMONES
i.
Endocrine hormones regulate a great variety of physiological processes.
ii.
Hormonal messages are specifically addressed.
1. The human endocrine system secretes a wide variety of hormones from the Endocrine gland that
enable the body to:
a. Maintain homeostasis (a steady state; e.g., insulin and glucagon maintain the blood glucose level
within rigid limits during feast or famine).
b. Respond to a wide variety of external stimuli (such as the preparation for “fight or flight” by
epinephrine and norepinephrine).
c. Follow various cyclic and developmental programs (for instance, sex hormones regulate sexual
differentiation, maturation, the menstrual cycle, and pregnancy).
1. Only those cells with a specific receptor for a given hormone will respond to its presence even though
nearly all cells in the body may be exposed to the hormone.
iii.
Hormone signals interact with target tissues by binding to receptors that transduce the signal to the
interior of the cell.
A. Pancreatic Islet Hormones Control Fuel Metabolism
i. The pancreatic hormones insulin and glucagon help control fuel metabolism.
1. The most potent physiological stimuli for the release of insulin and glucagon are, respectively, high
and low blood glucose concentrations.
2. Insulin stimulates muscle, liver, and adipose cells to store glucose for later use by synthesizing
glycogen, protein, and fat.
3. Glucagon, which is secreted in response to low blood glucose, has essentially the opposite effects:
a. It stimulates the liver to release glucose through the breakdown of glycogen (glycogenolysis) and the
synthesis of glucose (gluconeogenesis) from noncarbohydrate precursors.
b. It also stimulates adipose tissue to release fatty acids through lipolysis.
4. Somatostatin, which is also secreted by the hypothalamus, inhibits the release of insulin and
glucagon from islet cells.
B. Epinephrine and Norepinephrine Prepare the Body for Action
i. Catecholamines produced by the adrenal medulla bind to a- and b–adrenergic receptors on target
cells.
C. Steroid Hormones Regulate a Wide Variety of Metabolic and Sexual Processes
2
i.
Steroid hormones regulate fuel metabolism, salt and water balance, and sexual differentiation and
function.
D. Growth Hormone Binds to Receptors in Muscle, Bone, and Cartilage
i. Growth hormone exerts its effects by inducing dimerization of its receptor
2. RECEPTOR TYROSINE KINASES
A. Receptor Tyrosine Kinases Transmit Signals across the Cell Membrane
i. Autophosphorylation Activates Insulin Receptor
1. Insulin binds to receptor whose C-terminal domains have tyrosine kinase activity. Upon binding
insulin, the cytoplasmic protein tyrosine kinase domains get phosphorylated on specific Tyr residues.
This autophosphorylation activates the PTK so that it can phosphorylate other protein substrates.
B. Kinase Cascades Relay Signals to the Nucleus
i. Kinase cascades will then relay signals to their targets. The SH2-containing proteins that interact with
the insulin receptor substrates have varied functions. Some are kinases, some are phosphatases, and
some are GTPases that are therefore known as G proteins.
ii. When insulin activates the Ras signaling pathway, the result is an increase in protein synthesis that
supports cell growth and differentiation, a response consistent with insulin's function as a signal of
fuel abundance.
iii. How does a cell prevent inappropriate cross talk between closely related signaling pathways?
1. One way that this occurs is through the use of scaffold proteins that bind some or all of the
component protein kinases of a particular signaling cascade so as to ensure that the protein kinases of
a given pathway interact only with one another.
C. Some Receptors Are Associated with Non-Receptor Tyrosine Kinases
i. Ligand binding induces these tyrosine kinase–associated receptors to dimerize (and, in some cases, to
trimerize), often with different types of subunits, in a way that activates associated nonreceptor
tyrosine kinases (NRTKs).
D. Protein Phosphatases Are Signaling Proteins in Their Own Right
i. Intracellular signals must be “turned off” after the system has delivered its message so
that the system can transmit future messages. In the case of protein kinases, their activities are
balanced by the activities of protein phosphatases that hydrolyze the phosphoryl groups attached to
Ser, Thr, or Tyr side chains and thereby limit the effects of the signal that activated the kinase.
ii. Bubonic Plague (the flea-transmitted “Black Death”) is caused by the Yersinia bacteria.
1. Protein tyrosine phosphatases of this bacteria is far more catalytically active than other known
protein tyrosine phosphatases. Hence, when Yersinia injects this protein into a cell, the cell's phosphoTyr-containing proteins are catastrophically dephosphorylated.
2. Bacteria lack protein tyrosine kinases and hence do not synthesize phospho-Tyr residues, but it seems
like ancestral Yersinia acquired a protein tyrosine phosphatases gene from a eukaryote.
3. HETEROTRIMERIC G PROTEIN
A. G Protein–Coupled Receptors Contain Seven Transmembrane Helices
3
i.
Heterotrimeric G proteins are members of the superfamily of regulatory GTPases. Many G proteins
share common structural motifs that bind guanine nucleotides and catalyze the hydrolysis of GTP.
Many heterotrimeric G proteins participate in signal transduction systems that consist of three major
components:
1. G protein–coupled receptors
2. Heterotrimeric G proteins
3. Adenylate cyclase.
ii.
Activated Adenylate cyclase catalyzes the synthesis of cAMP from ATP.
iii.
The G protein–coupled receptors include:
1. In eukaryotic cells, its main target is protein kinase A (also known as cAMP-dependent protein kinase),
an enzyme that phosphorylates specific Ser or Thr residues of numerous cellular proteins.
a. cAMP is a second messenger, that is, it intracellularly transmits the signal originated by the
extracellular ligand.
1. Glucagon receptor, & a host of other proteins that bind peptide hormones, odorant (having an
odor) & tastant (having a taste) molecules, and eicosanoids.
iv.
The importance of these receptors is also evident in the fact that some 60% of the therapeutic drugs
presently in use target them.
B. Heterotrimeric G Proteins Dissociate on Activation
i.
The effect of G protein activation is short-lived, because Gα is also a GTPase that catalyzes the
hydrolysis of its bound GTP to GDP + Pi, although at the relatively sluggish rate of 2 to 3 min .
–1
ii.
When GTP is bound to Gα, its phosphate group promotes conformational changes causing Gα to
dissociate from Gβγ.
C. Adenylate Cyclase Synthesizes cAMP to Activate Protein Kinase A
i. In the absence of cAMP, protein kinase A is an inactive heterotetramer of 2 regulatory and 2 catalytic
subunits, R2C2.
1. The cAMP binds to the regulatory subunits to cause the dissociation of active catalytic monomers.
ii.
The targets of protein kinase A include enzymes involved in glycogen metabolism.
iii.
Each step of a signal transduction pathway can potentially be regulated.
1. For example, when epinephrine binds to the β-adrenoreceptor of a muscle cell, the sequential
activation of this system leads to the activation of glycogen phosphorylase, thereby making glucose-6phosphate available for glycolysis.
1. For example, the adenylate cyclase signaling pathway can be limited or reversed through ligand
activation of a receptor coupled to an inhibitory G protein.
a. The activity of the cAMP second messenger can be attenuated by the action of phosphodiesterases that
hydrolyze cAMP to AMP.
b. In addition, reactions catalyzed by protein kinase A are reversed by protein Ser/Thr phosphatases.
iv. Many drugs and toxins exert their effects by modifying components of the adenylate cyclase system.
D. Phosphodiesterases Limit Second Messenger Activity
4. THE PHOSPHOINOSITIDE PATHWAY
4
A. Ligand Binding Results in the Cytoplasmic Release of the Second Messengers IP3 and Ca2
i. Signal transduction via the phosphoinositide pathway generates the second messenger inositol
trisphosphate, which triggers Ca2 release, and diacylglycerol, which activates protein kinase C.
ii. Phosphoinositide pathway requires:
a. a receptor
b. a heterotrimeric G protein
c. a specific kinase
d. a phosphorylated glycerophospholipid that is a minor component of the plasma membrane.
1. It involves the production of 3 second messengers, inositol-1,4,5- trisphosphate (IP 3), Ca 2+ , and 1,2diacylglycerol (DAG).
B. Calmodulin Is a Ca2 -Activated Switch
i. In the presence of Ca2 , calmodulin binds and activates its target proteins.
1. The water-soluble IP 3 diffuses through the cytoplasm to the endoplasmic reticulum (ER).
2. There, it binds to and induces the opening of a Ca 2+ transport channel (an example of a receptor that
is also an ion channel), thereby allowing the efflux of Ca 2+ from the ER.
3. This causes the cytosolic [Ca 2+ ] to increase from ~0.1 μM to as much as 10 mM, which triggers such
diverse cellular processes as glucose mobilization and muscle contraction through the intermediacy of
the Ca 2+ -binding protein calmodulin (CAM).
4. ER contains embedded Ca 2+ –ATPases that actively pump Ca 2+ from the cytosol back into the ER so that
in the absence of IP , the cytosolic [Ca 2+ ] rapidly returns to its resting level.
3
C. DAG Is a Lipid-Soluble Second Messenger That Activates Protein Kinase C
i. PKC activation depends on lipid binding.
D. Epilog: Complex Systems Have Emergent Properties
i. A hormone can activate multiple signal transduction pathways to elicit a variety of intracellular
responses
5
CHAPTER 14
INTRODUCTION TO METABOLISM
1. OVERVIEW OF METABOLISM
A. Metabolism
i. Metabolic pathways are series of connected enzymatic reactions that produce specific products. Their
reactants, intermediates, and products are referred to as metabolites.
ii. Divided into 2 parts:
1. Catabolism, or degradation, in which nutrients and cell constituents are broken down to salvage their
components and/or to generate energy.
2. Anabolism, or biosynthesis, in which biomolecules are synthesized from simpler components.
iii. ATP and NADPH are the major free energy sources for biosynthetic reactions.
B. Cells Take Up the Products of Digestion
i. Human diet consists of 4 types of biomolecules
1. Proteins
2. Nucleic acids
ii.
Digestion reduces biomolecules to monomers
1. Amino acids
2. Nucleotides
3. Polysaccharides
4. Fats (particularly triacylglycerols)
3. Monosaccharides
4. Fatty acid
C. Nutrition Intake
1. Macronutrients proteins, carbohydrates, and lipids are broken down by the digestive system to their
component amino acids, monosaccharides, fatty acids, and glycerol which are then transported by the
circulatory system to the tissues. The metabolic utilization of the latter substances also requires the
intake of O 2 and water, as well as micronutrients composed of vitamins and minerals.
2. Mg 2+ is involved in nearly all reactions that involve ATP and other nucleotides, including the synthesis
of DNA, RNA, and proteins. Zn 2+ is a cofactor in a variety of enzymatic reactions including that catalyzed
by carbonic anhydrase. Ca 2+ , in addition to being the major mineral component of bones and teeth, is
a vital participant in signal transduction processes.
ii.
Metabolic reactions are catalyzed by enzymes. These reactions fall into 4 major types:
1.
2.
3.
4.
Oxidations & Reductions (catalyzed by oxidoreductases),
Group-transfer reactions (catalyzed by transferases and hydrolases)
Eliminations, Isomerizations, & Rearrangements (catalyzed by isomerases and mutases)
Reactions that Make or Break Carbon–Carbon Bonds (catalyzed by hydrolases, lyases, and ligases).
9 They, however, need cofactors to carry out their reactions.
2. CATABOLISM
i.
6
A striking characteristic of degradative metabolism is that the pathways for the catabolism of a large
number of diverse substances (carbohydrates, lipids, and proteins) converge on a few common
intermediates/
1. In many cases, a 2-carbon acetyl unit linked to coenzyme A to form acetyl-coenzyme A (acetylCoA).
ii.
This is followed by the oxidation of the acetyl carbons to CO2 by the citric acid cycle.
1. When one substance is oxidized (loses electrons), another must be reduced.
2. The citric acid cycle thus produces the reduced coenzymes NADH and FADH 2, which then pass their
electrons to O 2 to produce H 2O in the processes of electron transport and oxidative phosphorylation.
3. METABOLIC FUNCTIONS OF EUKARYOTIC ORGANELLES
i.
In multicellular organisms, compartmentation is carried a step further to the level of tissues and
organs.
1. The mammalian liver, for example, is largely responsible for the synthesis of glucose from
noncarbohydrate precursors so as to maintain a relatively constant level of glucose in the circulation,
whereas adipose tissue is specialized for storage of triacylglycerols.
4. METABOLIC FLUX MUST BE CONTROLLED
i.
ii.
Understanding the flux (rate of flow) of metabolites through a metabolic pathway requires knowledge
of which reactions are functioning near equilibrium and which are far from it.
Most enzymes in a metabolic pathway operate near equilibrium, however certain enzymes operate far
from equilibrium. This has several important implications:
1. Metabolic pathways are irreversible. A highly exergonic reaction (one with ΔG << 0) is irreversible;
that is, it goes to completion.
a. If such a reaction is part of a multistep pathway, it makes the entire pathway irreversible.
2. There is generally an irreversible (exergonic) reaction early in the pathway that “commits” its product
to continue down the pathway.
3. Catabolic and anabolic pathways differ.
a. If a metabolite is converted to another metabolite by an exergonic process, free energy must be supplied
to convert the second metabolite back to the first.
9 This energetically “uphill” process requires a different pathway for at least one of the
reaction steps.
A. Regulation Occurs at Steps with the Largest Free Energy Changes
i. Cells can regulate flux through a pathway by adjusting the rate of a reaction with a large free energy
change.
7
CHAPTER 15
GLUCOSE CATABOLISM
1. GLYCOLYSIS OVERVIEW
A. Glucose (a six-carbon molecule) is broken down into two 3-carbon molecules
Glucose + 2 NAD+ + 2ADP + 2Pi ® 2 pyruvate + 2NADH + 2ATP
B. Glycolysis occurs in 10 steps.
i. Steps 1-5 = energy investment
ii. Steps 6-10 = energy payoff
C. Electron carriers are reduced.
i. First 5 Steps of Glycolysis
ii. Last 5 Steps of Glycolysis
2. THE REACTIONS OF GLYCOLYSIS
A. First Stage of Glycolysis
i. Step 1: Hexokinase Reaction
1. Kinases are enzymes that phosphorylate
molecules.
2. ATP is invested; ATP hydrolysis drives the
reaction.
3. Reaction is irreversible
4. Hexokinase
a. Hexokinase is a ubiquitous, relatively nonspecific enzyme that catalyzes the phosphorylation of hexoses
such as D-glucose, D-mannose, and D-fructose.
b. 4 types of hexokinase isozymes are seen in mammals.
9 Isozymes = enzymes that catalyze the same reaction but are encoded in different genes.
c. Hexokinases I, II, and III have K m values of about 0.1 mM. Blood glucose concentration is 4 to 5 mM.
d.
e.
ii.
Glucokinase = hexokinase IV has a K m value of ~ 2-5 mM. (does not get inhibited by glucose 6phosphate and does not get saturated with glucose=works in high levels of glucose).
9 It is more abundant in liver and the insulin-secreting cells of pancreas.
9 In liver, the glucose 6-phosphate made by this enzyme is used for glycogen synthesis.
Muscle hexokinases I and II are allosterically inhibited by their product which happens at high cellular
concentration of glucose 6-phosphate.
Step 2: Phosphoglucose Isomerase Reaction
1. Conversion of a hexose to a pentose
2. Reaction is near equilibrium.
iii.
8
Step 3: Phosphofructokinase Reaction
1. Reaction is irreversible.
2. Another ATP is invested; ATP hydrolysis drives the reaction.
3. Glycolysis is regulated at this step.
4. Remember: Sugars can be in cyclic or linear forms
a. Cleavage of fructose-1,6-bisphosphate is easiest to understand
using the linear form of the molecule.
iv.
Step 4: Aldolase Reaction
v.
Step 5: Triose Phosphate Isomerase Reaction
1. The aldolase reaction is the reverse of an aldol condensation.
1. Converts DHAP to GAP (and vice versa)
2. Result: 2 GAP’s proceed through remainder of glycolysis
3. Even though ΔG>0, reaction proceeds forward because GAP is quickly consumed.
B. Second Stage of Glycolysis
i. Step 6: GAP Dehydrogenase Reaction
1. Notice: phosphate does not come from ATP.
2. NAD + is reduced to NADH.
3. Reaction is both a phosphorylation and an
oxidation-reduction reaction.
3-
4. Reaction is inhibited by AsO 4 , which
2-
competes with PO 4 for binding the
enzyme.
ii.
Step 7: Phosphoglycerate Kinase Reaction
iii.
Step 8: Phosphoglycerate Mutase Reaction
iv.
Step 9: Enolase Reaction Forms 2nd “High-Energy” Intermediate
v.
1. ATP is formed.
2. Since reaction occurs twice, 2 ATP have been
recouped from the energetic investment.
1. Phosphate gets moved to C ¾ 2.
1. Enolase catalyzes a dehydration reaction.
2. H 2 O is produced.
Step 10: Pyruvate Kinase Reaction
1. Final step of glycolysis
2. ATP formed: energetic payoff nets 2 ATP
3. 1 Glucose (a six-carbon molecule) has been broken down into 2 pyruvates (i.e., two three-carbon
molecules)
C. Standard & Physiological Free Energy Changes of Glycolytic Reactions
i. Identification of the rate-determining step(s) of the pathway by measuring the in vivo free energy
change for each reaction.
ii. Enzymes that operate far from equilibrium are potential control points.
3. FERMENTATION: THE ANAEROBIC FATE OF PYRUVATE
A. Under AEROBIC conditions, the pyruvate is completely oxidized via the citric acid cycle to CO2 and
H2O.
9
B. Under ANAEROBIC conditions, pyruvate must be converted to a reduced end product in order to
reoxidize the NADH produced by the GAPDH reaction. This occurs in two ways.
i. In muscle, pyruvate is reduced to lactate by lactate dehydrogenase (LDH) to regenerate NAD+ in a
process known as homolactic fermentation.
ii. In yeast, pyruvate is decarboxylated to yield CO and acetaldehyde, which is then reduced by NADH to
yield NAD+ and ethanol. This process is known as alcoholic fermentation.
2
1. The lactate can either be exported from the cell or converted back to pyruvate. Much of the lactate
produced in skeletal muscle cells is carried by the blood to the liver, where it is used to synthesize
glucose. The muscle fatigue and soreness it is due to the acidity of lactate.
C. Anaerobic fermentation results in the production of 2 ATP per glucose, whereas oxidative
phosphorylation yields up to 32 ATP per glucose.
i. However, the rate of ATP production by anaerobic glycolysis can be up to 100 times faster than that of
oxidative phosphorylation.
ii. Consequently, when tissues such as muscle are rapidly consuming ATP, they regenerate
it almost entirely by anaerobic glycolysis.
1. Homolactic fermentation does not really “waste” glucose since the lactate can be
aerobically reconverted to glucose by the liver.
4. REGULATION OF GLYCOLYSIS
A. Only 3 reactions of glycolysis:
1. Hexokinase
2. Phosphofructokinase
3. Pyruvate Kinase
i.
Those catalyzed by these reactions function with large
negative free energy changes in heart muscle under
physiological conditions.
ii.
These nonequilibrium reactions of glycolysis are candidates for flux-control points.
B. Phosphofructokinase Is the Major Flux-Controlling Enzyme of Glycolysis in Muscle
i. Phosphofructokinase plays a central role in control of glycolysis because it catalyzes
one of the pathway's rate-determining reactions.
ii. ATP acts as an allosteric inhibitor
iii. PFK is a tetrameric enzyme with 2 conformational states, R and T.
iv. In muscle, [ATP] is ~50 times greater than [AMP] and ~10 times greater than [ADP].
1. Consequently, a change in [ATP] from, for example, 1 to 0.9 mM, a 10% decrease, can
result in a 100% increase in [ADP] (from 0.1 to 0.2 mM) as a result of the adenylate
kinase reaction, and a >400% increase in [AMP] (from 0.02 to ~0.1 mM).
a. Therefore, a metabolic signal consisting of a decrease in [ATP]
too small to relieve PFK inhibition is amplified significantly
by the adenylate kinase reaction, which increases [AMP] by an
amount that produces a much larger increase in PFK activity.
9 The most potent allosteric effector of PFK is F2,6P
10
5. METABOLISM OF HEXOSES OTHER THAN GLUCOSE
A. Is Excess Fructose Harmful?
i. The consumption of fructose in the United States has increased at least 10-fold in the last quarter
century, in large part due to the use of high-fructose corn syrup as a sweetener in soft drinks and other
foods.
1. Fructose has a sweeter taste than sucrose and is inexpensive to produce.
ii.
One possible hazard of excessive fructose intake is that fructose catabolism in liver bypasses the PFKcatalyzed step of glycolysis and thereby avoids a major metabolic control point.
1. This could potentially disrupt fuel metabolism so that glycolytic flux is directed toward lipid
synthesis in the absence of a need for ATP production.
a. This hypothesis suggests a link between the increase in both fructose consumption and the recently
increasing incidence of obesity in the United States.
B. Fructose metabolism
i. In muscle, the conversion of fructose to the glycolytic intermediate F6P involves only 1 enzyme,
hexokinase.
ii. In liver (right), 7 enzymes participate in the conversion of fructose to glycolytic intermediates.
1. Liver contains a hexokinase homolog known as glucokinase, which phosphorylates only glucose
C. Galactose metabolism
i. Galactosemia is a genetic disease characterized by the inability to convert galactose to glucose. This
can lead to a buildup of toxic metabolic by-products.
1. For example, the increased galactose concentration in the blood results in a higher galactose
concentration in the lens of the eye, where the sugar is reduced to galactitol.
a. The presence of this sugar alcohol in the lens eventually causes cataract formation (clouding of the lens).
Galactosemia is treated by a galactose-free diet. Except for the mental retardation, this reverses all
symptoms of the disease.
6. THE PENTOSE PHOSPHATE PATHWAY
A. The pentose phosphate pathway is an oxidative pathway for producing NADPH and converting glucose
to ribose.
i. The reversible reactions of the pathway allow the interconversion of ribose and intermediates of
glycolysis and gluconeogenesis.
ii. Many endergonic reactions, notably the reductive biosynthesis of fatty acids and cholesterol, require
NADPH in addition to ATP.
1. Despite their close chemical resemblance, NADPH and NADH are not metabolically interchangeable.
a. NADH uses the free energy of metabolite oxidation to synthesize ATP (oxidative phosphorylation),
b. NADPH uses the free energy of metabolite oxidation for reductive biosynthesis.
2. This differentiation is possible because the dehydrogenases involved in oxidative and reductive
metabolism are highly specific for their respective coenzymes.
a. Indeed, cells normally maintain their [NAD+]/[NADH] ratio near 1000, which favors metabolite
oxidation, while keeping their [NADP+]/[NADPH] ratio near 0.01, which favors reductive
biosynthesis.
11
iii.
NADPH is generated by the oxidation of glucose-6-phosphate via an alternative pathway to glycolysis,
the pentose phosphate pathway (also called the hexose monophosphate shunt).
B. Pathway Reactions
i. Oxidative reactions of the pentose phosphate pathway produce NADPH.
1. Production of 6-phosphogluconate can
also occur in the absence of an enzyme.
ii.
The third step of the pentose phosphate
pathway involves oxidative decarboxylation.
iii. Ribose-5-phosphate is a precursor of the ribose unit of nucleotides.
C. Rearrangements of the products of the pentose phosphate pathway.
i. 3 of the five-carbon products of the oxidative phase of the pentose phosphate pathway are converted
to 2 fructose-6-phosphate (F6P) and 1 glyceraldehyde-3-Phosphate (GAP) by reversible reactions
involving: the transfer of two- and three-carbon units.
a. Each square represents a carbon atom in a monosaccharide.
2. This pathway also allows ribose carbons to be used in glycolysis and gluconeogenesis.
D. Relationship Between Glycolysis & Pentose Phosphate Pathway
E. Net Reaction for the Pentose Phosphate Pathway
glucose-6-phosphate + 2NADP++ H2O ® ribose-5-phosphate + 2NADPH + CO2+ 2H+
1. Ribose derivative is produced.
2. 2 NADPH molecules are formed.
3. Pathway is active in rapidly dividing cells.
12
CHAPTER 16
GLYCOGEN METABOLISM &
GLUCONEOGENESIS
1. GLYCOGEN BREAKDOWN
A. Storage Polysaccharides-Glycogen
i. Glycogen is a multibranched polysaccharide of glucose that serves as a form of energy storage in
animals and fungi.
1. The primary structure of glycogen resembles that of amylopectin, but glycogen is more highly
branched permitting the rapid mobilization of glucose in times of metabolic need.
2. Glycogen (in animals, fungi, and bacteria) and starch (in plants) can function to stockpile glucose for
later metabolic use.
a. In animals, a constant supply of glucose is essential for tissues such as the brain and red blood cells, which
depend almost entirely on glucose as an energy source (other tissues can also oxidize fatty acids for
energy.
b. The mobilization of glucose from glycogen stores, primarily in the liver, provides a constant supply of
glucose (~5 mM in blood) to all tissues.
9 When glucose is plentiful, such as immediately after a meal, glycogen synthesis accelerates. Yet
the liver's capacity to store glycogen is sufficient to supply the brain with glucose for about half a
day.
9 Under fasting conditions, most of the body's glucose needs are met by gluconeogenesis (literally,
new glucose synthesis) from noncarbohydrate precursors such as amino acids.
c. Not surprisingly, the regulation of glucose synthesis, storage, mobilization, and catabolism by glycolysis or
the pentose phosphate pathway is elaborate and is sensitive to the immediate and long-term energy
needs of the organism
3. The importance of glycogen for glucose storage is plainly illustrated by the effects of deficiencies of the
enzymes that release stored glucose.
a. McArdle's disease, is an inherited condition whose major symptom is painful muscle cramps on
exertion. The muscles in afflicted individuals lack the enzyme required for glycogen breakdown to yield
glucose. Although glycogen is synthesized normally, it cannot supply fuel for glycolysis to keep up with the
demand for ATP.
ii.
Glycogen is present in all cells but is most prevalent in skeletal muscle and in liver.
1. Glycogen granules are especially prominent in the cells that make the greatest use of glycogen:
a. muscle (up to 1–2% glycogen by weight) and liver cells (up to 10% glycogen by weight.
B. The Metabolic Uses of Glucose
i. Glucose-6-phosphate (G6P) is a key branch point and has several possible fates:
1. It can be used to synthesize glycogen
2. It can be catabolized via glycolysis to yield ATP and carbon atoms (as acetyl- CoA) that are further
oxidized by the citric acid cycle
13
3. It can be shunted through the pentose phosphate pathway to generate NADPH and/or ribose-5phosphate.
4. In the liver and kidney, G6P can be converted to glucose for export to other tissues via the
bloodstream.
C. Glycogen Phosphorylase Degrades Glycogen to Glucose-1-Phosphate
i. In the cell, glycogen is degraded for metabolic use by glycogen phosphorylase and glycogen
debranching enzyme.
ii. Glucose units are mobilized by their sequential removal from the nonreducing ends of glycogen (the
ends lacking a C1-OH group). Whereas glycogen has only 1 reducing end, there is a nonreducing end
on every branch.
1. Glycogen's highly branched structure therefore permits rapid glucose mobilization through the
simultaneous release of the glucose units at the end of every branch.
Glycogen (n residues) + Pi « Glycogen (n-1 residues) + G1P
1. Phosphorylase covalently binds the cofactor pyridoxal--5ʹ--phosphate which is a vitamin B 6 derivative.
The phosphorylase reaction mechanism which shows how PLP's phosphate group functions as a
general acid–base catalyst.
ii.
The phosphorylation of Ser 14 promotes phosphorylase's T (inactive) → R (active) conformational
change a manner that resembles allosteric control.
1. The T-state enzyme is inactive because it has a malformed active site and a surface loop that blocks
substrate access to its binding site.
a. The conformation of phosphorylase b is allosterically controlled by the effectors AMP, ATP, and G6P
and is mostly in the T state under physiological conditions.
b. In contrast, the phosphorylated form of the enzyme, phosphorylase a, is unresponsive to these
effectors and is mostly in the R state unless there is a high level of glucose.
2. Thus, under usual physiological conditions, the enzymatic activity of glycogen phosphorylase is largely
determined by its rates of phosphorylation and dephosphorylation
D. Glycogen Debranching Enzyme Acts as a Glucosyltransferase
i. Phosphorolysis proceeds along a glycogen branch until it approaches to within 4 or 5 residues of an
α(1→6) branch point, leaving a “limit branch.”
1. Glycogen debranching enzyme acts as an α(1→4) transglycosylase (glycosyltransferase) by
transferring an α(1→4)-linked trisaccharide unit from a limit branch of glycogen to the nonreducing
end of another branch.
2. This reaction forms a new α(1→4) linkage with 3 more units available for phosphorylase-catalyzed
phosphorolysis.
3. The α(1→6) bond linking the remaining glycosyl residue in the branch to the main chain is hydrolyzed
(not phosphorylyzed) by the same debranching enzyme to yield glucose and debranched glycogen.
a. About 10% of the residues in glycogen (those at the branch points) are therefore converted to glucose
rather than G1P. Debranching enzyme has separate active sites for the transferase and the
α(1→6)-glucosidase reactions which improves the efficiency of the debranching process.
E. Phosphoglucomutase Interconverts Glucose-1-Phosphate and Glucose-6-Phosphate
14
i.
ii.
A phosphoryl group is transferred from the active phosphoenzyme to G1P, forming glucose-1,6bisphosphate (G1,6P), which then rephosphorylates the enzyme to yield G6P
Glucose-6-Phosphatase Generates Glucose in the Liver
1. The G6P produced by glycogen breakdown can continue along the glycolytic pathway or the pentose
phosphate pathway. In the liver, G6P is also made available for use by other tissues.
a. Because G6P cannot pass through the cell membrane, it is first hydrolyzed by glucose-6-phosphatase
(G6Pase). G6Pase resides in the endoplasmic reticulum (ER) membrane.
b. Consequently, G6P must be imported into the ER by a G6P translocase before it can be hydrolyzed.
c. The resulting glucose and P i are then returned to the cytosol via specific transport proteins.
9 A defect in any of the components of this G6P hydrolysis system results in type I glycogen storage
disease.
d. Glucose leaves the liver cell via a specific glucose transporter named GLUT2 and is carried by the blood to
other tissues.
9 Muscle and other tissues lack G6Pase and therefore retain their G6P.
2. GLYCOGEN SYNTHESIS
i.
Glycogen synthesis and breakdown occur by separate pathways. The three enzymes that participate in
glycogen synthesis are:
1. UDP–Glucose Pyrophosphorylase
2. Glycogen Synthase
3. Glycogen Branching Enzyme.
ii.
The separation of synthetic and degradative pathways for glycogen was recognized by studying
McArdle's disease
A. UDP–Glucose Pyrophosphorylase Activates Glucosyl Units
i. The reaction catalyzed by UDP–glucose pyrophosphorylase
B. Glycogen Synthase Extends Glycogen Chains
i. Glycogenin Primes Glycogen Synthesis.
1. Glycogen synthase cannot simply link together 2 glucose residues; it can only extend an already
existing α(1→4)-linked glucan chain. How, then, is glycogen synthesis initiated?
a. In the first step of this process, a 349-residue protein named glycogenin acting as a glycosyltransferase,
attaches a glucose residue donated by UDPG to the OH group of its Tyr 194.
a. Glycogenin then extends the glucose chain by up to 7 additional UDPG-donated glucose residues to form
a glycogen “primer.”
b. Only at this point does glycogen synthase commence glycogen synthesis by extending the primer.
2. Analysis of glycogen granules suggests that each glycogen molecule is associated with only 1 molecule
each of glycogenin and glycogen synthase.
C. Glycogen Branching Enzyme Transfers Seven-Residue Glycogen Segments
i. Branching to form glycogen is accomplished by branching enzyme.
1. A branch is created by transferring a 7-residue segment from the end of a chain to the C6-OH group of
a glucose residue on the same or another glycogen chain.
2. Each transferred segment must come from a chain of at least 11 residues, and the new branch point
must be at least 4 residues away from other branch points
15
3. CONTROL OF GLYCOGEN METABOLISM
i.
The opposing processes of glycogen breakdown and synthesis are reciprocally regulated by allosteric
interactions and the covalent modification of key enzymes.
ii. Glycogen metabolism is ultimately under the control of hormones such as insulin, glucagon, and
epinephrine.
A. Glycogen Phosphorylase & Glycogen Synthase Are under Allosteric Control
i. Both glycogen phosphorylase and glycogen synthase are under allosteric control by effectors that
include ATP, G6P, and AMP.
ii. Muscle glycogen phosphorylase is activated by AMP and inhibited by ATP and G6P.
1. This suggests that when there is high demand for ATP (low [ATP], low [G6P], and high [AMP]), glycogen
phosphorylase is stimulated and glycogen synthase is inhibited, which favors glycogen breakdown.
2. Conversely, when [ATP] and [G6P] are high, glycogen synthesis is favored because glycogen synthase
is activated by G6P.
iii. Control by allosteric effectors is superimposed on control by covalent modification.
B. Glycogen Phosphorylase and Glycogen Synthase Undergo Control by Covalent Modification
C. Glycogen Metabolism Is Subject to Hormonal Control
9
i.
ii.
Glycogen metabolism in the liver is largely controlled by the polypeptide hormones insulin and glucagon
acting in opposition. In muscles and various tissues, control is exerted by insulin and by the adrenal
hormones epinephrine and norepinephrine.
Glucagon is released from the pancreas when the concentration of circulating glucose decreases to
less than ~5 mM, such as during exercise or several hours after a meal has been digested. Glucagon is
therefore critical for the liver's function in supplying glucose to tissues that depend primarily on
glycolysis for their energy needs. Muscle cells do not respond to glucagon because they lack the
appropriate receptor.
Epinephrine and norepinephrine, which are often called the “fight or flight” hormones, are released
into the bloodstream by the adrenal glands in response to stress. There are two types of receptors for
these hormones:
1. β-adrenoreceptor (β-adrenergic receptor), which is linked to the adenylate cyclase system.
a. Muscle cells, which have the β-adrenoreceptor, respond to epinephrine by breaking down glycogen for
glycolysis, thereby generating ATP and helping the muscles cope with the stress that triggered the
epinephrine release
2. α-adrenoreceptor (α-adrenergic receptor), whose second messenger causes intracellular [Ca 2+ ] to
increase.
a. Liver cells respond to epinephrine directly and indirectly because epinephrine promotes the release of
glucagon from the pancreas which activates glycogen phosphorylase and inactivates glycogen synthase.
iii.
16
Insulin and Epinephrine Are Antagonists
1. Insulin is released from the pancreas in response to high levels of circulating glucose (e.g.,
immediately after a meal).
2. Hormonal stimulation by insulin increases the rate of glucose transport into the many types of cells
that have both insulin receptors and insulin sensitive glucose transporters called GLUT4 on their
surfaces
9 e.g., muscle & fat cells, but not liver & brain cells.
In addition, [cAMP] decreases, causing glycogen metabolism to shift from glycogen breakdown to
glycogen synthesis by activating phosphoprotein phosphatase-1.
3. In liver, insulin stimulates glycogen synthesis as a result of the inhibition of glycogen synthase kinase.
a. This action decreases the phosphorylation of glycogen synthase, thus increasing its activity.
b. In addition, it is thought that glucose itself, may be a messenger to which glycogen metabolism system
responds. An increase in glucose concentration therefore promotes inactivation of glycogen phosphorylase
a through its conversion to phosphorylase b. The subsequent release of phosphoprotein phosphatase-1
activates glycogen synthase.
9 Thus, when glucose is plentiful, the liver can store the excess as glycogen.
b.
4. GLUCONEOGENESIS
i.
When dietary sources of glucose are not available and when the liver has exhausted its supply of
glycogen, glucose is synthesized from noncarbohydrate precursors by gluconeogenesis.
1. Gluconeogenesis provides a substantial fraction of the glucose produced in fasting humans, even
within a few hours of eating.
2. Gluconeogenesis occurs in liver and, to a lesser extent, in kidney.
iii.
The noncarbohydrate precursors that can be converted to glucose include the glycolysis products lactate
and pyruvate, citric acid cycle intermediates, and the carbon skeletons of most amino acids.
A. Pyruvate Is Converted to Phosphoenolpyruvate in Two Steps
i. The first unique reaction of gluconeogenesis
a. PEP Carboxykinase
1. Oxaloacetate is both a precursor for gluconeogenesis and an intermediate of the citric acid cycle.
b. When the citric acid cycle substrate acetyl-CoA accumulates, it allosterically activates pyruvate carboxylase,
thereby increasing the amount of oxaloacetate that can participate in the citric acid cycle.
9 Pyruvate carboxylase has a biotin prosthetic group
c. When citric acid cycle activity is low, oxaloacetate instead enters the gluconeogenic pathway.
ii.
Gluconeogenesis Requires Metabolite Transport between Mitochondria and Cytosol
1. The generation of oxaloacetate from pyruvate or citric acid cycle intermediates occurs only in the
mitochondrion. PEP or oxaloacetate are then transported across the mitochondrial membrane.
Oxaloacetate is transported by malate–aspartate shuttle system.
a. NADH is transported out of mitochondria
B. Gluconeogenesis and Glycolysis Are Independently Regulated
i. The second and third unique reactions of gluconeogenesis
1. Fructose-1,6-bisphosphate is hydrolyzed by fructose-1,6-bisphosphatase (FBPase).
1. Glucose-6-phosphate is hydrolyzed by glucose-6- phosphatase, the same enzyme that converts
glycogen-derived G6P to glucose and which is present only in liver and kidney.
a. The net energetic cost of converting 2 pyruvate molecules to one glucose molecule by gluconeogenesis
is 6 ATP equivalents which is less than the energetic profit (2 ATP) of converting 1 glucose molecule to 2
pyruvate molecules via glycolysis.
b. The energetic cost is paid to maintain the independent regulation of 2 opposing pathways.
ii.
17
Phosphofructokinase Is the Major Flux-Controlling Enzyme of Glycolysis in Muscle
1. Refer to chapter 15
iii.
Allosteric Effectors Influence Gluconeogenic Flux.
iv.
Regulation by Changing the amounts of enzymes synthesized.
1. Fructose-2,6-bisphosphate is an extremely potent allosteric activator of phosphofructokinase (PFK) and
an inhibitor of fructose-1,6-bisphosphatase (FBPase).
2. A bifunctional enzyme functions as both phosphofructokinase-2 (PFK-2) and fructose bisphosphatase-2
(FBPase-2).
a. For example, when [glucose] is low, glucagon stimulates the production of cAMP in liver cells. This
activates PKA to phosphorylate the bifunctional enzyme, which inactivates the enzyme's PFK-2 activity and
activates its FBPase-2 activity.
b. The net result is a decrease in [F2,6P], which shifts the balance between the PFK and FBPase reactions in
favor of FBP hydrolysis and hence increases gluconeogenic flux.
1. For example, insulin inhibits transcription of the gene for PEPCK, whereas high concentrations of
intracellular cAMP promote the transcription of the genes for PEPCK, FBPase, and glucose-6phosphatase, and repress transcription of the genes for glucokinase, PFK, and the PFK-2/FBPase-2
bifunctional enzyme.
18
CHAPTER 17
CITRIC ACID CYCLE
1. OVERVIEW OF THE CITRIC ACID CYCLE
A. Citric Acid Cycle or Krebs Cycle or the Tricarboxylic Acid (TCA) Cycle
i. The citric acid cycle oxidizes the acetyl group of acetyl-CoA to two molecules
of CO2 in a manner that conserves the liberated free energy in the reduced
compounds NADH and FADH2. The cycle is named after the product of its first
reaction, citrate.
1. One complete round of the cycle yields 2 molecules of CO 2, 3 NADH, 1 FADH 2,
and 1 high- energy compound (GTP or ATP).
ii.
The circular pathway oxidizes acetyl groups from many sources, not just
pyruvate. Because it accounts for the major portion of carbohydrate, fatty acid, and amino acid
oxidation, the citric acid cycle is often considered the “hub” of cellular metabolism.
iii. The citric acid cycle is a central pathway for recovering energy from several metabolic fuels, including
carbohydrates, fatty acids, and amino acids, that are broken down to acetyl-CoA for oxidation.
B. General Features of the Citric Acid Cycle
i. The net reaction of the citric acid cycle is:
3 NAD+ + FAD + GDP + Pi + acetyl-CoA ® 3 NADH + 2
FADH2 + GTP + CoA + 2CO2
ii. The oxaloacetate that is consumed in the first step of the citric
acid cycle is regenerated in the last step of the cycle. Thus, the
citric acid cycle acts as a multistep catalyst that can oxidize an
unlimited number of acetyl groups.
iii. In eukaryotes, all the enzymes of the citric acid cycle are
located in the mitochondria, so all substrates, including NAD
and GDP, must be generated in the mitochondria or be
transported into mitochondria from the cytosol. Similarly, all
the products of the citric acid cycle must be consumed in the
mitochondria or transported into the cytosol
iv. The carbon atoms of the 2 molecules of CO2 produced in one
round of the cycle are not the 2 carbons of the acetyl group that
began the round. These acetyl carbon atoms are lost in
subsequent rounds of the cycle.
1. However, the net effect of each round of the cycle is the oxidation of 1 acetyl group to 2 CO 2 .
v.
19
Citric acid cycle intermediates are precursors for the biosynthesis of other compounds (e.g.,
oxaloacetate for gluconeogenesis).
2. SYNTHESIS OF ACETYL-COENZYME A
A. Pyruvate Dehydrogenase is a Multienzyme Complex
i. Acetyl-CoA is formed from pyruvate through oxidative decarboxylation by a multienzyme complex
named pyruvate dehydrogenase.
1. This complex contains multiple copies of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl
transacetylase (E2), and dihydrolipoyl dehydrogenase (E3).
2. The pyruvate dehydrogenase complex is a ~10,000-kD particle
ii.
Advantages of multienzymes:
1. The distance that substrates must diffuse between active sites is minimized, thereby enhancing the
reaction rate.
2. The channeling of metabolic intermediates between successive enzymes in a metabolic pathway
reduces the opportunity for these intermediates to react with other molecules, thereby minimizing
side reactions.
3. The reactions catalyzed by a multienzyme complex can be coordinately controlled.
iii.
Coenzymes and Prosthetic Groups of Pyruvate Dehydrogenase
1. Thiamine Pyrophosphate (TPP)
2. Lipoic Acid
3. Coenzyme A (CoA)
4. FAD
+
5. NAD
B. The Pyruvate Dehydrogenase Complex Catalyzes Five Reactions
1. E1 catalyzes
reaction 1 and 2
i.
2. E2 catalyzes
reaction 3
Step 1: Decarboxylation of Pyruvate
1. E1 decarboxylates pyruvate; CO 2 is
released
2. E2 uses a lipoamide cofactor
3. Disulfide bond = active portion
ii.
Step 2: Transfer from E1 to E2
iii.
Step 3: Acetyl Transfer to CoA
iv.
Step 4: Restoration of E2
v.
Step 5: Restoration of E3
1. A leftover hydroxyethyl group is
transferred to E2.
1. An acetyl group is transferred to CoA.
1. E2 is restored
2. E3 uses a disulfide bond and an FAD cofactor
3. Lipoamide is oxidized by E3’s disulfide
1. E3 is restored to its oxidized state and NADH is produced
2. Electrons move to FAD to form FADH2.
+
3. Electrons move from FADH2 to NAD + to form NADH and H
3. ENZYMES OF THE CITRIC ACID CYCLE
A. Reaction 1: Citrate Synthase Adds an Acetyl Group to Oxaloacetate
20
3. E3 catalyzes
reaction 4 and 5
B. Reaction 2: Aconitase Isomerizes Citrate to Isocitrate
i. Aconitase contains a [4Fe–4S] iron–sulfur cluster that presumably
coordinates the OH group of citrate to facilitate its elimination.
Iron–sulfur clusters normally participate in redox processes;
Aconitase is an intriguing exception.
C. Reaction 3: Isocitrate Dehydrogenase Releases the First CO2
D. Reaction 4: α-Ketoglutarate Dehydrogenase Releases the Second
CO2
ii. The α-ketoglutarate dehydrogenase reaction chemically
resembles the reaction catalyzed by the pyruvate dehydrogenase
multienzyme complex.
i. Fates of Carbons in the Citric Acid Cycle
1. The two carbons released in the 1st round of the citric acid cycle did not come from acetyl-CoA.
E.
F.
G.
H.
I.
Reaction 5: Succinyl-CoA Synthetase Catalyzes Substrate Level Phosphorylation
Reaction 6: Succinate Dehydrogenase Generates Ubiquinol
Reaction 7: Fumarase Catalyzes a Hydration Reaction
Reaction 8: Malate Dehydrogenase Regenerates Oxaloacetate
Products of Citric Acid Cycle
i. The citric acid cycle is an energy-generating cycle
1. 1 NADH = 2.5 ATP
2. 1 FADH 2 = 1.5 ATP
a. The electrons carried by NADH and FADH 2 are funneled into the electrontransport chain, which culminates with the reduction of O 2 to H 2O.
a. The energy of electron transport is conserved in the synthesis of ATP by
oxidative phosphorylation.
4. REGULATION OF CITRIC ACID CYCLE
A. Pyruvate Dehydrogenase Is Regulated by Product Inhibition and Covalent Modification
i. Product inhibition by NADH and acetyl-CoA.
Pyruvate + CoA + NAD+ ® acetyl-CoA + CO2 + NADH
ii. Covalent modification by phosphorylation/dephosphorylation of E1.
1. In eukaryotes, the products of the pyruvate dehydrogenase reaction, NADH and acetyl-CoA, also
activate the pyruvate dehydrogenase kinase resulting in phosphorylation of a specific dehydrogenase
Ser residue inactivates the pyruvate dehydrogenase complex.
2. Insulin, the hormone that signals fuel abundance, reverses the inactivation by activating pyruvate
dehydrogenase phosphatase, which removes the phosphate groups from pyruvate dehydrogenase.
a. Recall that insulin also activates glycogen synthesis by activating phosphoprotein phosphatase. Thus, in
response to increases in blood [glucose], insulin promotes the synthesis of acetyl-CoA as well as glycogen.
B. Three Enzymes Control the Rate of the Citric Acid Cycle
21
i.
There is no single flux-control point in the citric acid cycle; rather, flux control is distributed among
several enzymes. The flux is controlled primarily by three simple mechanisms:
1. Substrate Availability,
2. Product Inhibition
3. Competitive Feedback Inhibition by intermediates further along the cycle.
ii.
Perhaps the most crucial regulators of the citric acid cycle are:
1. Its Substrates
2. Acetyl-CoA and Oxaloacetate
3. Its product, NADH.
5. REACTIONS RELATED TO THE CITRIC ACID CYCLE
A. Other Pathways Use Citric Acid Cycle Intermediates
i. Gluconeogenesis: Since the citric acid cycle is a cyclical pathway, any of its intermediates can be
converted to oxaloacetate and used for gluconeogenesis.
ii. Fatty acid biosynthesis is a cytosolic process that requires acetyl-CoA. Acetyl-CoA is generated in the
mitochondrion but transported across the mitochondrial membrane as citrate.
ATP + citrate + CoA ® ADP + Pi + oxaloacetate + acetyl-CoA
B. Some Reactions Replenish Citric Acid Cycle Intermediates
i. An increase in the concentrations of citric acid cycle intermediates supports increased flux of acetyl
groups through the cycle.
1. For example, flux through the citric acid cycle may increase as much as 60- to 100-fold in muscle cells
during intense exercise.
a. Pyruvate carboxylase senses the need for more citric acid cycle intermediates through its activator, acetylCoA.
9 This activates pyruvate carboxylase, which replenishes oxaloacetate.
b. Pyruvate can also accept an amino group from glutamate to generate alanine and the citric acid cycle
intermediate α-ketoglutarate.
2. Both of these mechanisms help the citric acid cycle efficiently catabolize the acetyl groups derived—also
from pyruvate—by the reactions of the pyruvate dehydrogenase complex.
C. The Glyoxylate Cycle Shares Some Steps with the Citric Acid Cycle
i. Plants, bacteria, and fungi, but not animals, possess enzymes that mediate the net conversion of acetylCoA to oxaloacetate, which can be used for gluconeogenesis.
1. The glyoxylate cycle results in the net conversion of 2 acetyl-CoA to succinate instead of to 4
molecules of CO 2 as would occur in the citric acid cycle.
ii.
Some human pathogens use the glyoxylate cycle, sometimes to great advantage.
1. For example, Mycobacterium tuberculosis, which causes tuberculosis, can persist for years in the
lung without being attacked by the immune system.
a. During this period, the bacterium subsists largely on lipids, using the citric acid cycle to produce
precursors for amino acid synthesis and using the glyoxylate cycle to produce carbohydrate precursors.
2. Drugs that are designed to inhibit the bacterial isocitrate lyase can therefore potentially limit the
pathogen's survival.
22
CHAPTER 18
ELECTRON TRANSPORT &
OXIDATIVE PHOSPHORYLATION
The electrons from the Citric Acid Cycle pass into the mitochondrial electron-transport chain, a system of linked
electron carriers.
ELECTRON TRANSPORT PROCESS EVENTS:
•
By transferring their electrons to other substances, the NADH and FADH2 are reoxidized to NAD and FAD so that they can
•
participate in additional substrate oxidation reactions.
The transferred electrons participate in the sequential oxidation– reduction of multiple redox centers (groups that
undergo oxidation– reduction reactions) in 4 enzyme complexes before reducing O2 to H2O.
•
During electron transfer, protons are expelled from the mitochondrion, producing a proton gradient across the
mitochondrial membrane. The free energy stored in this electrochemical gradient drives the synthesis of ATP from ADP
and Pi through oxidative phosphorylation.
1. THE MITOCHONDRION
A. Mitochondria Contain a Highly Folded Inner Membrane
i. The mitochondrion is the site of eukaryotic oxidative metabolism.
ii. A eukaryotic cell typically contains ~2000 mitochondria.
iii. A mitochondrion is bounded by a smooth outer membrane and contains a highly folded inner
membrane.
1. Mitochondria contains: pyruvate dehydrogenase, the citric acid cycle enzymes, the enzymes catalyzing
fatty acid oxidation, and the enzymes and redox proteins involved in electron transport and oxidative
phosphorylation.
2. The proteins mediating electron transport and oxidative phosphorylation are bound in the inner
mitochondrial membrane.
3. The matrix is a gel-like solution that contains extremely high concentrations of the soluble enzymes of
oxidative metabolism as well as substrates, nucleotide cofactors, inorganic ions, DNA, RNA, and
ribosomes.
↳
It is therefore with good reason that the mitochondrion is often described as the cell's “power plant.”
B. Ions and Metabolites Enter Mitochondria via Transporters
i. Unlike the outer membrane, the inner membrane, which is ~75% protein by mass, is not permeable
to most ions and metabolites . It is freely permeable only to O2, CO2, and H2O and contains, numerous
transport proteins then control the passage of metabolites such as ATP, ADP, pyruvate, Ca2+, and
phosphate.
ii. NADH Transport
1. Glycerophosphate Shuttle
a. The electrons of cytosolic NADH are transported to the mitochondrial electron-transport chain in 3 steps
23
9
9
9
(1) Cytosolic oxidation of NADH by dihydroxyacetone phosphate catalyzed by 3-phosphoglycerol
dehydrogenase.
(2) Oxidatio of 3-phosphoglycerol by flavoprotein dehydrogenase with reduction of FAD to FADH
(3) Reoxidation of FADH 2 with passage of electrons into the electron-transport chain.
2. Malate–Aspartate Shuttle
a. The malate-aspartate shuttle transports reducing agents across the inner mitochondrial membrane
iii.
A different transport system is used to move ATP from the matrix
to the cytosol.
A. ATP translocase protein imports ADP and exports ATP.
B. A symport protein permits simultaneous movement of
P i and H
+.
2. ELECTRON TRANSPORT
A. Electron Transport Is an Exergonic Process
i. The coupling of NADH oxidation to ATP synthesis is achieved by an electron-transport chain in which
electrons pass through 3 protein complexes.
1. The thermodynamic efficiency is thought to be ~70% while in comparison, the energy efficiency of a
typical automobile engine is <30%.
B. Electron Carriers Operate in Sequence
i. Electrons are carried from Complexes I and II to Complex III by coenzyme Q (CoQ or ubiquinone; so
named because of its ubiquity in respiring organisms), and from Complex III to Complex IV by the
peripheral membrane protein cytochrome c.
1. Complex I catalyzes oxidation of NADH by CoQ
2. Complex III catalyzes oxidation of CoQ (reduced) by Cytochrome C
3. Complex IV catalyzes oxidation of reduced Cytochrome C by O 2 , the terminal electron acceptor of
the Electron Transport Process
4. Complex II catalyzes oxidation of FADH 2 by CoQ
↳
24
This redox reaction does not release sufficient free energy to synthesize ATP; it functions only to inject the
electrons from FADH2 into the electron-transport chain.
ii.
Inhibitors Reveal the Workings of the Electron Transport Chain.
iii.
Reduction Potentials of ETC Components
1. Rotenone, a plant toxin used by Amazonian Indians to poison fish, also used as an insecticide, blocks
electron transport in Complex I.
2. Antimycin A is an antibiotic which blocks electron transport in Complex III.
3. Cyanide blocks electron transport in Complex IV.
C. Electron Transport
i. Electron donated by NADH and FADH2 travel
through this chain from lower to higher standard
reduction potentials.
ii. The transit of electrons in each complex occurs by
a stepwise mechanism, according to the
reduction potential of their various redox centers.
iii. The spatial arrangement of the groups indicates the likely path of the electrons.
a.
iv.
Note that redox centers do not need to come into contact in order to transfer an electron.
An electron's quantum mechanical properties enable it to quickly “tunnel” (jump) between proteinembedded redox groups that are separated by less than ~14 Å.
1. Because electron transfer rates decrease exponentially with the distance between redox centers (they
exhibit an ~10-fold decrease for each 1.7 Å increase in distance), electron transfers over distances
longer than ~14 Å always involve chains of redox centers.
D. Complex I Accepts Electrons from NADH
i. Complex I (NADH-coenzyme Q oxioreductase) passes electrons from NADH to CoQ, and may be the
largest protein complex in the inner mitochondrial membrane
+
NADH + CoQ(oxidized) ® NAD + CoQ(reduced)
i.
Complex I Contains Multiple Coenzymes.
1. FMN and CoQ can each adopt 3 oxidation states. They are capable of accepting and donating either 1
or 2 electrons because their semiquinone forms are stable.
a. Quinones such as ubiquinone and plastoquinone are lipid-soluble electron and proton carriers.
2. Iron–sulfur clusters can undergo 1-electron oxidation and reduction and thus can have oxidation
states between the normal Fe 2+ and Fe 3+
a. Iron-sulfur and hemes are tightly bound 1-electron carriers in proteins
3. FMN is tightly bound to proteins; however, CoQ has a hydrophobic tail that makes it soluble in the
inner mitochondrial membrane's lipid bilayer.
b. FMN, and FAD are water-soluble coenzymes but usually bound to enzymes, called flavoproteins.
4. FMN and CoQ, which can transfer 1 or 2 electrons at a time, therefore provide an electron conduit
between the 2-electron donor NADH and the 1-electron acceptors, the cytochromes.
a. NAD and NADP are water soluble capable of moving from one enzyme to another.
ii.
Complex I Translocates Four Protons.
1. Electron transfer and proton flow in complex I.
2. The eukaryotic form contains an FMN-containing flavoprotein and at least 8 Fe-S centers.
E. Complex II Contributes Electrons to Coenzyme Q
i. Complex II or succinate–coenzyme Q oxidoreductase contains the citric acid cycle enzyme succinate
dehydrogenase, passes electrons from succinate to CoQ.
1. The free energy for electron transfer from succinate to CoQ is insufficient to drive ATP synthesis. The
complex is nevertheless important because it allows relatively high- potential electrons to enter the
electron- transport chain by bypassing Complex I.
ii.
25
Electron Transfer in Complex II
1. Note that Complexes I and II, despite their names, do not operate in series. But both accomplish the
same result: the transfer of electrons to CoQ from reduced substrates (NADH or succinate).
a. CoQ, which diffuses in the lipid bilayer among the respiratory complexes, therefore serves as a sort of
collection point for electrons.
F. Cytochromes are Electron-Transport Heme Proteins
i. Cytochromes are redox-active proteins which contain heme groups that alternate between their Fe(II)
and Fe(III) oxidation states during electron transport.
ii. Within each group of cytochromes, different heme group environments may be characterized by
slightly different α peak wavelengths.
1. For this reason, it is convenient to identify cytochromes by the wavelength (in nm) at which its α band
absorbance is maximal (e.g., cytochrome b560 in Complex II).
iii.
Each group of cytochromes contains a differently substituted heme group coordinated with the redoxactive iron atom.
1. The b-type cytochromes contain protoporphyrin IX, which also occurs in myoglobin and hemoglobin.
2. The heme group of c-type cytochromes differs from protoporphyrin IX in that its vinyl groups have
added Cys sulfhydryls across their double bonds to form thioether linkages to the protein.
G. Complex III Translocates Protons via the Q Cycle
i. Complex III (also known as coenzyme Q–cytochrome c oxidoreductase or cytochrome bc1) passes
electrons from reduced CoQ to cytochrome c.
1. It contains 2 b-type cytochromes, 1 cytochrome c1, and one [2Fe–2S] cluster in which one of the Fe
atoms is coordinated by two His residues rather than two Cys residues (and which is known as a Rieske
center after its discoverer, John Rieske).
ii.
The electrons that flow to cytochrome c1 are transferred to cytochrome c, which, unlike the other
cytochromes of the respiratory electron-transport chain, is a soluble protein.
1. It shuttles electrons between Complexes III and IV on the outer surface of the inner mitochondrial
membrane.
iii.
The circuitous route of electrons from Ubiquinol to cytochrome c is described by the Q cycle.
1. Results of the Q Cycle
a. 2 electrons from QH2 reduce 2 molecules of cytochrome c.
b. 4 protons are pumped into the intermembrane space.
9 2 from QH 2 in the first round
9
2 from QH 2 in the second round
H. Complex IV Reduces Oxygen to Water
i. Cytochrome c oxidase (Complex IV) catalyzes the 1-electron oxidations of 4 consecutive reduced
cytochrome c molecules and the concomitant 4- electron reduction of 1 O2 molecule:
ii.
4 Cytochrome c (Fe2+) + 4 H++ O2 ® 4 Cytochrome c (Fe3+) + 2 H2O
A Proposed Model for Cytochrome c Oxidase Activity
1. O 2 is reduced to H 2 O via the Fe-S clusters in cytochrome c oxidase.
2. For every 2 electrons donated by cytochrome c, 2 protons are translocated to the intermembrane space
3. 2 protons from the matrix are also consumed in the reaction: ½ O 2 → H 2 O
26
3. OXIDATIVE PHOSPHORYLATION
Key Concepts
R Proton translocation drives the rotation of a portion of ATP synthase.
R Rotation-induced conformational changes allow ATP synthase to bind ADP and Pi to phosphorylate ADP, and to
release ATP.
R Because ATP synthesis is indirectly linked to electron transport, the P:O ratio is not a whole number.
R The supply of reduced cofactors determines the rate of oxidative phosphorylation.
A. Chemiosmotic Theory Links Electron Transport to Oxidative Phosphorylation
i. Chemiosmotic Theory states: the free energy of electron transport is conserved by pumping H from
the mitochondrial matrix to the intermembrane space to create an electrochemical H gradient across the
inner mitochondrial membrane. The electrochemical potential of this gradient is harnessed to
synthesize ATP.
ii. Electron Transport causes a Proton Gradient
1. Electron transport causes Complexes I, III, and IV to transport protons across the inner mitochondrial
membrane from the matrix, a region of low [H + ], to the intermembrane space, a region of high [H + ].
2. The free energy change of transporting a charged H + from one side of the membrane to the other has a
chemical as well as an electrical component.
a. In a liver mitochondrion, the pH of its matrix is 0.75 units higher than that of its intermembrane space.
9 ΔG for proton transport out of this mitochondrial matrix is therefore 21.5 kJ · mol –1 .
b. An ATP molecule's estimated physiological free energy of synthesis, around +40 to +50 kJ · mol –1, is too
large for ATP synthesis to be driven by the passage of a single proton back into the mitochondrial matrix;
9 2-3 protons are required.
3. Since the pH outside the mitochondrion (side 2) is less than the pH of the matrix (side 1), the export of
protons from the mitochondrial matrix (against the proton gradient) is an endergonic process.
B. ATP Synthase is Driven by the Flow of Protons
i. ATP synthase (Complex V): The protein that taps the electrochemical proton gradient to
phosphorylate ADP.
ii. ATP synthase, also known as proton-pumping ATP synthase and F1F0–ATPase
1. It is a multisubunit transmembrane protein composed of two functional units, F 0 and F 1
a.
b.
iii.
ATP Synthase Function
iv.
ATP synthase rotates as it translocates protons.
1. Use the electrochemical gradient to drive phosphorylation.
2. Form ATP
1.
2.
3.
4.
27
F 0 is a water-insoluble transmembrane protein while
F 1 is a water-soluble peripheral membrane protein.
9 F 1 is easily and reversibly dissociated from F 0 by treatment with urea.
9 Solubilized F 1 hydrolyzes ATP but cannot synthesize it (hence the name ATPase).
+
H binds to a c subunit.
The c subunit moves away from the a subunit.
As a new c subunit reaches the a subunit a proton is released.
One rotation of the ring translocates 8 protons
v.
The 3 αβ pairs interact asymmetrically with the γ subunit.
vi.
The binding change mechanism explains how ATP is made.
1. The F1 component of mitochondrial ATP synthase has 5 types of subunit with composition α3β3γδε
2. Apparently the γ subunit, which rotates within the fixed α3β3 assembly, acts as a molecular camshaft
in linking the proton gradient–driven F0 engine to the conformational changes in the catalytic sites of
F1
1. The αβ subunits form three different
conformations.
a. O = open
b. T = tight
c. L = loose
2. ADP and Pi bind to the loose
conformation.
3. ATP is formed in the tight
conformation.
4. ATP is released in the open
conformation
C. The P/O Ratio Relates to the Amount of ATP Synthesized to the Amount of Oxygen Reduced
i. ATP synthesis is tightly coupled to the proton gradient; that is, ATP synthesis re- quires the discharge of
the proton gradient, and the proton gradient cannot be discharged without the synthesis of ATP.
1. Electrons that enter the electron-transport chain as NADH generate 10 protons enough to drive the
9
ii.
synthesis of ~3 ATP through the F 1 F 0 –ATPase.
2. Electrons that enter the electron- transport chain as FADH 2 at Complex II bypass Complex I and
therefore lead to the transmembrane movement of only 6 protons, enough to synthesize ~2 ATP.
These stoichiometric relationships are called P/O ratios.
In actively respiring mitochondria, P/O ratios are almost certainly not integral numbers.
1. The proton gradient is dissipated to some extent by the nonspecific leakage of protons back into the
matrix and by the consumption of protons for other purposes, such as the transport of P i into the
matrix.
a. Taking P i transport into account the P/O ratios above are actually 2.5 ATP/NADH and 1.5 ATP/FADH 2.
9 Oxidation of 1 glucose therefore gives 32 ATP.
9 2.5 ATP/NADH × 10 NADH/glucose + 1.5 ATP/FADH2 × 2 FADH2/glucose + 2 GTP/glucose from the Citric
Acid Cycle + 2 ATP/glucose from glycolysis = 32 ATP/glucose.
D. Oxidative Phosphorylation Can Be Uncoupled from Electron Transport
i. Electron transport (the oxidation of NADH and FADH2 by O2) and oxidative phosphorylation (the proton
gradient–driven synthesis of ATP) are normally tightly coupled.
ii. Electron transport (the oxidation of NADH and FADH2 by O2) and oxidative phosphorylation (the proton
gradient–driven synthesis of ATP) are normally tightly coupled. Compounds such as 2,4-dinitrophenol
(DNP) have been found to “uncouple” electron transport and ATP synthesis because it increases the
permeability of the inner mitochondrial membrane to H+. Under physiological conditions, the
dissipation of an electrochemical H+ gradient, which is generated by electron transport and uncoupled
from ATP synthesis, produces heat.
1. Consequently, in the 1920s, DNP was used as a “diet pill,” a practice that was effective in inducing
weight loss but often caused fatal side effects.
iii.
Uncoupling in Brown Adipose Tissue Generates Heat
1. Newborn mammals that lack fur, such as humans, as well as hibernating mammals, all contain brown
fat in their neck and upper back that generates heat by nonshivering thermogenesis. Other
sources of heat are the ATP hydrolysis that occurs during muscle contraction (in shivering or any other
movement).
a. The mechanism of heat generation in brown fat involves the regulated uncoupling of oxidative
phosphorylation. Brown fat mitochondria contain a proton channel known as uncoupling protein (also
called thermogenin).
4. CONTROL OF OXIDATIVE PHOSPHORYLATION
R The activities of the pathways that produce ATP are under strict coordinated control so that ATP is never produced
more rapidly than necessary.
A. The Rate of Oxidative Phosphorylation Depends on the ATP and NADH Concentrations
i. Pathways of Oxidative Metabolism Are Coordinately Controlled.
1. The few irreversible reactions constitute potential control points of the pathways and usually are
catalyzed by regulatory enzymes that are under allosteric control.
a. In the case of oxidative phosphorylation, the pathway from NADH to Cytochrome c functions near
equilibrium. However, the Cytochrome c oxidase reaction (the terminal step of the electron- transport
chain) is irreversible and is therefore a potential control site.
9 Cytochrome c oxidase, appears to be controlled primarily by the availability of reduced
Cytochrome c.
9 The concentration of reduced Cyt. c ultimately depends on the intramitochondrial ratios of
[NADH]/[NAD+] and [ATP]/[ADP][Pi] (also known as the ATP mass action ratio).
b. For example in an individual at rest, the rate of ATP hydrolysis to ADP and P i is minimal and the ATP mass
action ratio is high The concentration of reduced cytochrome c is therefore low and the rate of oxidative
phosphorylation is minimal.
2. Mitochondria contain an 84-residue protein, called IF 1 that functions to regulate ATP synthase.
a. In actively respiring mitochondria, in which the matrix pH is relatively high, IF 1 exists as an inactive
tetramer. However, below pH 6.5, the protein dissociates into dimers and in this form, inhibits the ATPase
activity of the F 1 component by binding to the interface between its α and β subunits to trap the ATP
bound.
9 This appears to be a mechanism to prevent ATP hydrolysis when respiratory activity is temporarily
interrupted by lack of O 2 .
Otherwise, the F1F0–ATPase would reverse its direction of rotation as driven by the hydrolysis of ATP (then
generated by glycolysis), thus depriving the cell of its remaining energy resources.
3. One particularly interesting regulatory effect is the inhibition of phosphofructokinase (PFK) by citrate.
9
a. When demand for ATP decreases, [ATP] increases and [ADP] decreases.
b. Because isocitrate dehydrogenase is activated by ADP and α-ketoglutarate dehydrogenase is inhibited by
c.
ATP, the citric acid cycle slows down.
This causes the citrate concentration to build up. Citrate leaves the mitochondrion via a specific transport
system and, once in the cytosol, acts to restrain further carbohydrate breakdown by inhibiting PFK.
B. Aerobic Metabolism Has Some Disadvantages
i. What happens during oxygen deprivation?
1. Rapidly respiring tissues, such as the heart and brain, are particularly susceptible to damage by
oxygen deprivation.
a. Consider 2 common causes of human death: heart attack and stroke.
29
In the absence of O 2 , a cell, which must then rely only on glycolysis for ATP production, rapidly
depletes its stores of phosphocreatine and glycogen.
9 As the rate of ATP production falls below the level required by membrane ion pumps for
maintaining proper intracellular ion concentrations, osmotic balance is disrupted so that the cell
and its membrane-enveloped organelles begin to swell.
9 The resulting overstretched membranes become permeable, thereby leaking their enclosed
contents.
For this reason, a useful diagnostic criterion for myocardial infarction is the presence in the blood of heartspecific enzymes, such as the H-type isozyme of lactate dehydrogenase.
9
b.
30
ii.
Partial Oxygen Reduction Produces Reactive Oxygen Species (ROS).
iii.
Cells are Equipped with Antioxidant Mechanisms.
1. Although the 4-electron reduction of O 2 by Cytochrome c oxidase is nearly always orchestrated with
great rapidity and precision, O 2 is occasionally only partially reduced, yielding oxygen species that
readily react with a variety of cellular components.
2. All classes of biological molecules are susceptible to oxidative damage caused by free radicals that
includes polyunsaturated lipids, DNA and enzymes.
a. Such observations have led to the free- radical theory of aging, which holds that free-radical reactions
arising during the course of normal oxidative metabolism are at least partially responsible for the aging
process.
1. Superoxide Dismutase enzyme
2. Catalase enzyme
3. Glutathione Peroxidase enzyme