Download 3.1 METABOLIC PATHWAYS §3.1a Overview of

Part III => METABOLISM and ENERGY
§3.1 METABOLIC PATHWAYS
§3.1a Overview of Metabolism
§3.1b High-Energy Compounds
§3.1c Redox Reactions
Section 3.1a:
Overview of Metabolism
Synopsis 3.1a
- Metabolism is the process through which living organisms
break down nutrients (or synthesize biomolecules) to generate
energy needed to drive various biochemical processes needed
for the maintenance of life—ie the sum of all chemical
reactions in a living organism!
- In metabolic processes, nutrients can be broken down to
generate energy (catabolism), or biomolecules can be
synthesized from simpler chemicals (anabolism)
- Metabolic processes usually occur via a series of enzymecatalyzed reactions that are collectively referred to as a
“metabolic pathway”
- Nutrition (dietary intake) can be divided into two major
categories:
1) Macronutrients  Proteins/carbohydrates/lipids
2) Micronutrients  Vitamins/minerals
Characteristics of Vitamins (Organic Molecules)
- Vitamins are small organic molecules that organisms are unable to
synthesize and thus must be obtained from diet
- Vitamins are essential for driving a multitude of chemical reactions
- Vitamins can be divided into two groups: water-soluble and fat-soluble
(Hydrophilic)
(Lipophilic)
(intestinal)
Major Minerals & Trace Elements (Inorganic Compounds)
(mM)
(nM)
- Minerals constitute naturally-occurring native elements and their compounds (inorganic)—eg
metal ions, oxides, sulfides, and halides
- Like vitamins, minerals play a key role in driving a wide variety of cellular processes and enzymecatalyzed reactions
- Minerals that are required in high concentration (in the mM range) are called “Major Minerals”
- Minerals that are required in low concentration (in the nM range) are called “Trace Elements”
Catabolic & Anabolic Pathways are Coupled!
- Metabolic pathways are a series of
connected enzymatic reactions
- Reactants, intermediates and
products of such reactions are
referred to as “metabolites”
- In catabolic pathways (degradation),
the breakdown of nutrients results
in the release of free energy—which
is conserved either via synthesis of
ATP from ADP, or reduction of
NAD(P)+ to NAD(P)H
- In anabolic pathways (biosynthesis),
ATP and NAD(P)H serve as major
energy sources or “currencies” for
the synthesis of biomolecules
Overview of Catabolism
- Catabolism of macronutrients
(eg proteins, polysaccharides
and triglycerides) usually
converges on acetyl-CoA—
where CoA is coenzyme A
- Acetyl-CoA is then further
catabolized via the citric acid
cycle to generate NADH and
FADH2
- NADH and FADH2 are
subsequently oxidized via
oxidative phosphorylation to
generate additional sources
of free energy
Metabolic Functions of Eukaryotic Organelles
Metabolic pathways occur in specific cellular compartments—eg glycolysis occurs
in the cytosol, whereas citric acid cycle is carried out in the mitochondrion
Organ-Specific Sources of Energy
Major organs are specialized in their ability to
generate energy from only specific metabolites:
Organ-Specific Metabolic Profiles
Kidneys
- Glucose
Brain
- Glucose (primary)
- Ketone bodies (starvation)
Liver
- Amino acids!!
Adipose Tissue
- Glucose
- Fatty acids
Heart
Skeletal Muscle
- Glucose
- Fatty acids
- Ketone bodies
Heart Muscle
- Fatty acids
- Ketone bodies
Erythrocyte
Erythrocytes (no mitochondria!)
- Carry all but use none—O2!
- Meet their own energy needs via anaerobic glycolysis—
oxidation of glucose to lactate via fermentation (§3.2)
Major Characteristics of Metabolic Pathways
(1) Metabolic pathways are irreversible
- While most enzymatically-driven steps in a metabolic pathway operate near equilibrium
(∆G ≈ 0), metabolic pathways usually harbor one or more exergonic steps that operate far
from equilibrium (∆G << 0)
- Such thermodynamic behavior imparts directionality on metabolic pathways
(2) Metabolic pathways harbor a committed step
- Metabolic pathways are usually characterized by an exergonic step (∆G << 0) early in the
pathway that “commits” the pathway to continue downstream
(3) Catabolic and anabolic pathways differ
- Given that the conversion of metabolite 1 to
metabolite 2 via intermediate A is exergonic
(∆G << 0), the same route cannot be used for
the synthesis of metabolite 1 from metabolite
2 due to unfavorable thermodynamics
- Rather, a separate pathway must be employed to convert metabolite 2 back to metabolite
1 via intermediates X and Y
- Existence of such independent pathways is an important property of metabolic pathways
as it allows independent control of degradation versus biosynthesis
Metabolic Flux
- Living organisms are thermodynamically open systems that tend to maintain a
steady-state rather than reach equilibrium—doing so would equate to death!
- “Steady-state” implies that the rates of synthesis and degradation of metabolic
intermediates within a cell are more or less equal such that their concentrations
change little over time—eg the blood glucose level is maintained @ 1mg/ml!
- The rate of flow of metabolites through a metabolic pathway is referred to as
“metabolic flux”—it is determined by the slowest or the rate-determining step in
a metabolic pathway
- The metabolic flux of a pathway can be altered or modulated depending on the
needs of the cell and/or organism
- This is usually achieved by altering the rate of the rate-determining step in a
metabolic path via one of the following mechanisms:
(1) Allosteric Control
(2) Covalent Modification
(3) Substrate Cycling
(4) Gene Expression
(1) Metabolic Flux: Allosteric Control
- Many enzymes involved in controlling metabolic pathways are allosterically regulated
by effectors—such as substrates and products resulting from within or other
metabolic steps as well as coenzymes
- In allosteric regulation, effectors modulate proteins by virtue of their ability to bind in
an uncompetitive manner to a site other than the ligand binding pocket or enzyme
active site
- In what has come to be known as the “negative feedback regulation”, the final
product of a pathway inhibits an earlier step so as to overhaul the metabolic flux
(2) Metabolic Flux: Covalent Modification
- Many enzymes involved in controlling metabolic pathways are subject to posttranslational modifications (PTMs) such as phosphorylation
- Phosphorylation on residues such as serine, threonine, and/or tyrosine can enhance
or inhibit the enzyme activity
- Such PTM is ultimately controlled by extracellular signals such as hormones
(3) Metabolic Flux: Substrate Cycling
- In many metabolic pathways, a forward reaction is
opposed by a reverse step—eg consider the overall
conversion of substrate A into product D via
intermediates B and C
- In what is referred to as “substrate cycle”, enzyme f
converts B to C but enzyme r does the opposite
- Thus, enzymes f and r essentially act in a concerted
manner to cycle B to C and back again—hence
substrate cycling!
- Substrate cycling provides an highly sensitive
regulatory mechanism in that the overall metabolic
flux can be allosterically modulated not only via
enhancement of enzymatic activity of f but also by
inhibition of enzymatic activity of r and vice versa!
- Substrate cycling thus renders metabolic pathways more
sensitive to the concentrations of allosteric effectors than would
otherwise be possible via regulation of a unidirectional step
(4) Metabolic Flux: Gene Expression
- In response to metabolic needs, the cellular concentrations of many enzymes
involved in metabolic pathways can be modulated via gene expression, thereby
enhancing metabolic flux
Exercise 3.1a
- List the categories of macronutrients and micronutrients required for
mammalian metabolism and provide examples of each
- Explain the roles of ATP and NADPH in catabolic and anabolic
reactions
- Give some reasons why enzymes are essential for the operation of
metabolic pathways
- Explain the metabolic significance of reactions that function near
equilibrium and reactions that function far from equilibrium
- Discuss the mechanisms by which the flux through a metabolic
pathway can be controlled. Which mechanisms can rapidly alter flux?
Section 3.1b:
High-Energy Compounds
Synopsis 3.1b
- Organisms capture the free energy released on degradation of
nutrients in the form of “high-energy” compounds such as ATP
- Subsequent breakdown of ATP is used to power otherwise
endergonic reactions
- The “high-energy” of ATP is related to the large negative free
energy change associated with the hydrolysis of its
phosphoanhydride bonds
- ATP hydrolysis can be coupled to an endergonic reaction such that
the net reaction is favorable
Need for Energy Currency
+ Energy (ATP)
∆Go = -2850 kJ/mol
- Combustion of fuel (or oxidation of food) such as glucose is highly exergonic as it
releases considerable amount of free energy (-2850 kJ/mol)
- How can such energy be harnessed to drive endergonic reactions?
- How about storing this released energy into “high-energy” compounds—or simply,
energy reservoirs/carriers/wallets—as a form of energy “currency” to be used later
to drive endergonic processes?
- Indeed, the cellular machinery couples the energy released from the oxidation of
food to the synthesis of energy currencies such as ATP and NAD(P)H
- ATP and NAD(P)H can then be used to “pay for” cellular processes that are otherwise
thermodynamically unfavorable!
Hydrolysis of “High-Energy” Compounds
- Being referred to as “high-energy”
compounds is highly misleading and an
unfortunate misnomer
- Although the hydrolysis of “high-energy”
compounds releases large amounts of
free energy, such release of energy does
not result from the breaking of their
“high-energy” bonds
- On the contrary, bond-breaking is an
endergonic not exergonic process—ie it
consumes rather than releases energy!
- In essence, the “high-energy”
compounds harbor “unstable” bonds
that can be easily hydrolyzed into
thermodynamically more stable (lower
energy) products!!
- So what makes the hydrolysis of “highenergy” compounds such as ATP an
highly exergonic process?!
Why Is ATP Hydrolysis So Exergonic?!
Highly exergonic hydrolysis (-31 kJ/mol) of
the terminal phosphoanhydride bond of ATP
into ADP and inorganic phosphate (Pi) is due
to three major factors:
(1) Lower electrostatic repulsion—ATP hydrolysis
results in the minimization of electrostatic
repulsions between the negatively charged
O atoms of phosphoanhydride bonds
(2) Higher solvation energy—The products of hydrolysis
(eg ADP and Pi) are better solvated than ATP
due to more favorable hydrogen bonding
interactions with water, thereby resulting in
greater (more favorable) solvation energy
(3) Greater resonance stabilization—Since the electrons on terminal O atoms
are more delocalized than the bridging phosphoanhydride O atoms,
hydrolysis of the terminal phosphoanhydride bond of ATP replaces one
bridging oxygen atom with two new terminal oxygen atoms resulting in
greater electronic stabilization of ADP and Pi relative to ATP
Inorganic
Phosphate (Pi)
Being a “high-energy” compound, ATP serves as the cell’s primary energy currency!
ATP Hydrolysis Coupled to Synthesis of Glucose-6-P
- Being a “high-energy compound”, ATP can spontaneously transfer its terminal phosphoryl
group (-PO32-) to other compounds with lower phosphate hydrolysis potential—eg glucose
- Phosphorylation of glucose to glucose-6-phosphate (glucose-6-P) is an endergonic process
- Coupling this endergonic process to exergonic hydrolysis of ATP easily overcomes the
thermodynamic barrier with an overall ∆G << 0
- The half-reactions shown above are purely hypothetical and do not actually occur!
- Rather, hexokinase catalyzes the transfer of the terminal phosphoryl group (-PO32-) from
ATP directly to glucose to generate glucose-6-P (the first step of glycolysis—see §3.2)
PEP Hydrolysis Coupled to Synthesis of ATP
(PEP)
- Phosphorylation of ADP into ATP is an endergonic process
- Coupling this endergonic process to exergonic hydrolysis of phosphoenolpyruvate (PEP)
renders it thermodynamically feasible with an overall ∆G << 0
- The half-reactions shown above do not actually occur!
- Rather, pyruvate kinase catalyzes the transfer of the phosphoryl group (-PO32-) from PEP
directly to ADP to generate ATP (the final step of glycolysis—see §3.2)—a process referred
to as “substrate-level phosphorylation” due to the fact that the phosphoryl group is
transferred to ADP from another compound or substrate (eg PEP) as opposed to the energy
stored in the transmembrane chemical potential (proton) gradient utilized in “oxidative
phosphorylation”—see §3.6
Flow of Phosphoryl Groups to and from ATP
Glycolytic
intermediates
Energy reservoir in skeletal
muscle and brain
Phosphoryl groups flow
from the “high-energy”
donors to “low-energy”
acceptors
The squiggle (~) denotes
a “high-energy”
phosphate bond!
Exercise 3.1b
- What kinds of molecules do cells use as
energy currency?
- Why is ATP a “high-energy” compound?
- Describe the ways an exergonic process can
drive an endergonic process
Section 3.1c:
Redox Reactions
Synopsis 3.1c
- Redox reactions involve the transfer of electrons between
compounds—ie they couple reduction and oxidation
- The free energy of electrons is captured via generation of reduced
compounds and released upon their oxidation—release of such
free energy is needed to drive many metabolic reactions
- The tendency of an oxidized compound to gain electrons (or
become reduced) is rationalized in terms of the so-called
reduction potential (ε)—the higher the ε, the greater the
tendency to be reduced!
- Free energy and reduction potential are negatively related—the
greater the reduction potential difference (+∆ε) between two
compounds, the more negative the free energy change (-∆G)
Oxidizing and Reducing Agents
Reducing agent (electron donor)—loses electrons (or hydrogen) and becomes oxidized
(its oxidation state increases!)—eg Fe2+ loses an electron and becomes oxidized to Fe3+
Oxidizing agent (electron acceptor)—gains electrons (or hydrogen) and becomes reduced
(its oxidation state decreases!)—eg Fe3+ gains an electron and becomes reduced to Fe2+
Redox Half-Reactions
- Consider the following redox reaction:
Fe3+ + Cu+ <=> Fe2+ + Cu2+
which occurs during the oxidation of
cytochrome c oxidase in the mitochondrion
- In the context of an electrochemical cell—a
device capable of generating electrical energy
from chemical reactions—the above redox
reaction can be divided into two half-reactions:
Cu+
<=> Cu2+ + eFe3+ + e- <=> Fe2+
K+
Iron half-cell
KNO3
NO3-
Copper half-cell
- Simply put, the copper half-cell undergoes oxidation by liberating electrons (electron-rich),
while the iron half-cell experiences reduction by consuming electrons (electron-deficient)
- Such redox pair of half-reactions thus sets up an “electron gradient” across the two half-cells
resulting in an electrical potential difference or electromotive force (∆ε) that drives the flow
of electrons from electron-rich half-cell (copper) to electron-deficient half-cell (iron) through
an external circuit (eg a conducting wire)—how can we measure ∆ε?
- A salt bridge (eg a soaked filter paper in an electrolyte such as KNO3) is necessary to prevent
the build-up of charge difference across the two half-cells by enabling the flow of ions (eg K+
toward the iron half-cell, and NO3- toward the copper half-cell) to maintain electronic
neutrality—failure to do so will impede the flow of electrons between the half-cells!
Reduction Potential Difference (∆ε)
- Consider the generalized redox reaction:
A + B+ <=> A+ + B
where
------ ∆ε = εox - εred => ∆ε = εB - εA
+ = Indicative of the oxidized state of corresponding species
εA = Reduction potential of half-reaction of species A (vide infra)
εB = Reduction potential of half-reaction of species B (vide infra)
∆ε = Reduction potential difference between oxidizing (ox) and
reducing (red) agents—ie species B and A, respectively
- The half-reactions can be expressed as follows (electrons flow from A to B):
A
<=> A+ + e------εA
B+ + e- <=> B
------εB
- The reduction potential (ε) is a measure of the tendency of a species to undergo
reduction (or to gain electrons)—the higher the value (more positive) of ε, the
greater the reduction tendency
- Thus, εB > εA, since species B (acting as an oxidizing agent) displays higher tendency
than species A to undergo reduction
- How can we relate the reduction potential difference (∆ε) to the standard reduction
potential difference (∆ε°) and free energy change (∆G)?
Nernst Equation
- ∆ε is related to ∆ε° by the so-called Nernst equation:
∆ε = ∆ε° + [RT/zF]lnQ
where
[1]
∆ε = Reduction potential difference (V)
∆ε° = Standard reduction potential difference (V)
R = Universal molar gas constant (1.99 cal/mol/K, or 8.32 J/mol/K)
T = Absolute temperature (K)
z = Number of electrons transferred in the redox reaction
F = Faraday constant (96,485 C/mol, or 96,485 J/V/mol) => 1C=1J/V
Q = Reaction quotient (similar to equilibrium constant)
- Q is defined as:
Q = [Aox][Bred] / [Ared][Box]
[2]
- ∆ε is related to the free energy change (∆G) released by the redox reaction as
follows:
∆G = -zF∆ε
[3]
- Thus, a positive ∆ε indicates a spontaneous redox reaction and vice versa
Standard Reduction Potentials (ε°)
- Half-reactions with large positive ε° are
strong electron acceptors (oxidizing agents)
and energetically favorable
- Half-reactions with large negative ε° are
strong electron donors (reducing agents)
- Electrons spontaneously pass from halfreactions with lower ε° to increasing ε°
- Thus, both NADH and FADH2 serve as
electron donors to reduce O2 to H2O during
oxidative phosphorylation—the free energy
released is harnessed for ATP synthesis
- For example, free energy (∆G°) released by
the reduction of O2 by NADH via the
following redox reaction is:
0.5O2 + NADH + H+ <=> H2O + NAD+
∆G° = -zF∆ε°
where ∆ε° = (+0.815V) - (-0.315V) = 1.13V
F = 96,485 J/V/mol
z=2
thus ∆G° = -(2).(96,485 J/V/mol).(1.13V)
=> ∆G° = -218 kJ/mol
Redox Players
Major redox players, or cofactors, involved in mediating electron
transfer from reduced metabolites to other compounds include:
(1) Nicotinamide adenine dinucleotide (NAD+)
(2) Flavin adenine dinucleotide (FAD)
(3) Flavin mononucleotide (FMN)
(4) Coenzyme Q (CoQ)
(5) Heme (Haem in Imperial English)
(6) Iron-sulfur clusters (Fe-S)
Redox Players: NAD+
STRUCTURE
Nicotinamide
REDUCTION
Ribose
Phosphoanhydride
bond
Ribose
- Only the nicotinamide moiety of
NAD+ serves as the site of reversible
reduction
Adenine
phosphorylated
in NADP
Nicotinamide adenine dinucleotide (NAD+)
- NAD+ adopts only two oxidation
states (oxidized NAD+ and reduced
NADH)—implying that it can only
accept a pair of electrons as
opposed to an unpaired electron
Redox Players: FAD
STRUCTURE
REDUCTION
Phosphoanhydride bond
AMP
Ribose
Flavin
Ribitol is the reduced
form of ribose—cf aldose
vs alditol (§1.2)
Flavin adenine dinucleotide (FAD)
Unlike NAD, FAD can adopt three oxidation
states (oxidized FAD, radical FADH. and
reduced FADH2)—it can accept both paired
and unpaired electrons!
Redox Players: FMN
STRUCTURE
Phosphoester
bond
REDUCTION
Flavin
Flavin mononucleotide (FMN)
FMN differs from FAD in that it lacks AMP!
Like FAD, FMN can also adopt three oxidation
states (oxidized FMN, radical FMNH. and
reduced FMNH2)—it can accept both paired
and unpaired electrons!
Redox Players: CoQ
STRUCTURE
REDUCTION
n
where n = 6-10
Coenzyme Q (CoQ)
also known as ubiquinone
(lipophilic)
Like FAD and FMN, CoQ also adopts three
oxidation states (oxidized CoQ, radical CoQH.
and reduced CoQH2)—it can accept both
paired and unpaired electrons!
Redox Players: Heme
- Heme is comprised of an heterocyclic ring called porphyrin with an iron ion at its
center—the redox properties of heme are largely owed to the ability of iron to undergo
transition between an oxidized (Fe3+) and reduced (Fe2+) state
- Heme exists in several biologically important forms such as heme a, heme b and heme c
- Heme occurs as a co-factor in a wide variety of proteins such as Mb and Hb (heme a—
see §2.4) and cytochromes a/b/c (hemes a/b/c—see §3.6)
- By virtue of the ability of iron to transition between an oxidized (Fe3+) and reduced
(Fe2+) state, heme plays a key role in orchestrating a plethora of redox reactions
Redox Players: Fe-S Clusters
- Iron-sulfur (Fe-S) clusters are comprised of iron ions
bridged between sulfide ions and further
coordinated by either cysteine or cysteine/histidine
sidechain groups within protein chains
- In iron-sulfur proteins, the two most commonly
occurring Fe-S clusters are [2Fe-2S] and [4Fe-4S]
- In [2Fe-2S] cluster, two iron ions are bridged
between two sulfide ions—and each iron ion is
further coordinated to either two CYS or CYS/HIS
residues to adopt a tetrahedral geometry
- In [4Fe-4S] cluster, four iron ions are bridged
between four sulfide ions carving out a cube-like
structure—and each iron ions is further coordinated
to a CYS or HIS to adopt a tetrahedral geometry
- By virtue of the ability of iron to transition between
an oxidized (Fe3+) and reduced (Fe2+) state, the Fe-S
clusters play a key role in orchestrating the transfer
of electrons from reduced to oxidized compounds
Exercise 3.1c
- What are the metabolic roles of the coenzymes NAD+ and FAD?
- Explain why NADH and FADH2 are a type of energy currency in the cell
- Explain the terms of the Nernst equation
- When two half-reactions are combined, how can you predict which
compound will be oxidized and which will be reduced?
- How is ∆ε related to ∆G?