1
Chapter 13
Takusagawa’s Note©
Chapter 13: Introduction to Metabolism
1. METABOLIC PATHWAYS
1. Metabolic pathways are irreversible.
A
1
2
Y
Metabolism of proteins, carbohydrates and
lipids produces ATP (energy currency),
CO2 and H2O.
X
An example is shown in Fig. 15-2.
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Chapter 13
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Takusagawa’s Note©
2. Every metabolic pathway has a first committed step.
This step is generally an irreversible (exergonic) reaction (ΔG < 0).
3. All metabolic pathways are regulated.
The first committed step is often its rate-limiting step, which is regulated by the enzymes that
catalyze their first committed steps.
4. Metabolic pathways in eukaryotic cells occur in specific cellular locations.
e.g., ATP is generated in the mitochondria but utilized in the cytosol.
2. BIOORGANIC REACTION MECHANISMS
Four categories:
1. Group-transfer reactions
2. Oxidations and reductions
3. Eliminations, isomerizations, and rearrangements
4. Reaction that make or break C-C bonds
A. Chemical logic
- Homolytic and heterolytic bond cleavage.
- For C-H bond, the electron pair remains with the carbon atom is the predominant mode
(heterolytic).
-
-
Nucleophiles are negatively charged or contain unshared electron pair that easily form
covalent bonds with electron-deficient centers (electrophiles).
Nucleophile attack --- The electron-deficit centers are attacked by the electron pairs of
nucleophiles.
Nucleophilic groups are listed in Fig. 15-5.
1. Amino group
2. Hydroxyl group
3. Imidazole group
4. Sulfhydryl group
Electrophiles that contain an electron-deficient atom.
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Chapter 13
3
B. Group-transfer reactions
Y: + A-X → Y-A + X:
- The most common transferred groups (Fig. 15-7):
1. Acyl groups
2. Phosphoryl groups
3. Glycosyl groups.
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Takusagawa’s Note©
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Chapter 13
C. Oxidations and reductions
accompany a C-H bond cleavage with the ultimate loss of two bonding electrons of the
carbon atom. These electrons are transferred to an electron acceptor such as NAD+.
H
R
B +
O
C
H O C H +
NH2
+
N
R'
R"
H O
C
H
R
B H +
O C
+
R'
NH2
N
R"
D. Eliminations, isomerizations, and rearrangements
Elimination reactions to form C=C bonds from C-C single bonds by dehydration
- There are three mechanisms (Fig. 15-9a)
1. Concerted reactions with the simultaneous elimination of OH- and H+.
2. Stepwise reactions with the C-O bond breaking first to form a carbocation (acid catalysis).
3. Stepwise reactions with the C-H bond breaking first to form a carbanion (base catalysis).
-
Dehydration reactions catalyzed by enzymes are either (2) acid catalysis or (3) base catalysis.
Trans (anti) eliminations are the most prevalent biochemical mechanism. Cis (syn)
eliminations are less common.
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Chapter 13
E. Biochemical isomerizations
- involve intramolecular hydrogen atom shifts so as to change the location of a double bond.
F. Rearrangement reactions
- break and reform C-C bonds so as to rearrange a molecule’s carbon skeleton.
e.g., L-methylmalonyl-CoA ↔ succinyl-CoA
H
H
COO-
C
C
H
H
C
S
methylmalonylCoA mutase
CoA
CoA
H
COO-
H
C
C
S
C
H
O
H
O
L-Methylmalonyl-CoA
Succinyl-CoA
G. Reactions that make and break C-C bonds
- form the basis of both degradative and biosynthetic metabolism.
- Elimination of H+ or CO2 produces a carbanion (C:), which nucleophilic attacks a
electrophilic carbonyl carbon atom.
C
-
+
C
O
C
C
OH
e.g., breakdown of glucose to CO2 involves in five such cleavages while its synthesis
involves in the reverse process.
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Chapter 13
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Takusagawa’s Note©
3. EXPERIMENTAL APPROACHES TO THE STUDY OF METABOLISM
A. Metabolic inhibitors, growth studies and biochemical genetics
1. Metabolic inhibitors
E
E1
E
- A simple metabolic pathway: A ⎯ ⎯
→ B ⎯ ⎯2 → C ⎯ ⎯3 → D
- The precursor A and the product D are known, but the intermediates B and C are unknown.
- If the E2 activity is inhibited by an inhibitor, the intermediate-B will be accumulated and thus
be detectable.
- Example: Iodoacetate inhibits irreversibly enzymes which contains Cys residues in the active
sites.
- When iodoacetate is added in glycolysis, it causes accumulation of fructose-1,6bisphosphate, indicating that fructose-1,6-bisphosphate is one of the intermediates of
glycolysis.
- Genetic defects also cause metabolic intermediates to accumulate. For example (bad example),
- Phenylketouria is resulted in lacking of the enzyme that catalyzes Phe → Tyr.
- The secondary pathway converts Phe → Phenylpyruvate.
- This result leads originally into a misinterpretation.
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Chapter 13
2. Growth studies
- X-ray irradiation produces various mutant bacteria, some of them require a specific nutrient
for growth. These mutants are called auxotrophic mutants.
- For example: Pathway of arginine biosynthesis
- The three autotropic mutants 1, 2 and 3, which were isolated after X-ray irradiation did not
grow under a normal condition, but they grow if ornithine and/or citrulline and/or arginine
are added into the culture media as shown below.
Mutant-1
Mutant-2
Mutant-3
-
With ornithine
grow
not grow
not grow
With citrulline
grow
grow
not grow
With arginine
grow
grow
grow
This study indicates that
1. Arginine is synthesized from → ornithine → citrulline → arginine.
2. Three different enzymes are involved.
3. Enzymes are specified by genes since X-ray radiation gives damages on DNA.
Use of isotopes
- We can label the specific molecules by isotopes without changing their chemical properties.
- The metabolites containing the isotopes can be detected by various methods, such as mass
spectroscopy and NMR.
- If the radioactive isotopes are used, the metabolites are detected by proportional counting,
liquid scintillation counting and autoradiography.
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Chapter 13
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8
See half-life (t1/2)of some trace isotopes
in Table 15-1.
ln 2 0.693
t1 / 2 =
=
, where k = rate
k
k
constant for the decay process.
Precursors of biosynthesis
- Example: Precursors of heme
- The possible precursor molecules of
heme are ammonia, Gly, Glu, Pro, Leu.
- The nutrients containing the 15N-labeled
these possible precursors are fed to rats.
- The heme groups are isolated from the
rats and are analyzed.
- The heme contains 15N when 15Gly is fed
to rats, indicating that Gly is the
metabolic origin of the N-atoms in
heme.
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Chapter 13
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Takusagawa’s Note©
Biosynthesis order
- The tails of phospholipids contain vinyl ether and ether. There are two possible pathways
for biosynthesis of ether- and vinyl ether-containing phospholipids.
-
Starting materials are radioactively labeled. Then the radioactivities of ether- and vinyl
ether-products are monitored.
If A is precursor of B (stating material*→ A* →B* → later product*), then the radioactive
monitoring curve should be one shown in Fig. 15-19.
Because the [A] reaches its maximum faster than the [B] reaches its maximum, and as the
[A] decreases the [B] reaches its maximum.
For the phospholipid biosynthesis, the ether-containing lipids reach its maximum faster than
the vinyl ether-containing lipids reach its
maximum, indicating that the ethercontaining lipids are precursors. Thus,
the pathway of the phospholipid
biosynthesis is Scheme II in Fig. 15-18.
Isolated organs, cells, and subcellular
organelles
- Metabolic pathways in eukaryotic cells
occur in specific cellular locations.
- Those are determined by using isotope
labeling. For example, oxidative
phosphorylation occurs in the
mitochondria whereas glycolysis and
fatty acid biosynthesis occur in the
cytosol.
- A complete list is given in Table 15-2.
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Chapter 13
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Takusagawa’s Note©
4. THERMODYNAMICS OF PHOSPHATE COMPOUNDS
Endergonic processes (ΔG > 0) --- require energy to carry out.
Exergonic processes (ΔG < 0)--- produce energy.
ATP (adenosine triphosphate)
- is the “high-energy” intermediate that constitutes the most common cellular energy currency.
- consists of an adenine moiety and three phosphoryl groups.
- linked via a phosphoester bond followed by two phosphoanhydride bonds (high energy bonds).
A. Phosphoryl-transfer reactions
R1-O-PO32- + R2-OH ←⎯→ R1-OH + R2-O-PO32- Some of the most important reactions of ATP synthesis and hydrolysis are:
ATP + H2O ←⎯→ ADP + Pi
ATP + H2O ←⎯→ AMP + PPi
Pi = orthophosphate (PO43-)
PPi = pyrophosphate (P2O74-)
-
ATP hydrolyses are highly exergonic
reaction. Thus, numerous endergonic
biochemical processes are coupled with
ATP hydrolysis so as to drive them to
complete.
- ATP has an intermediate phosphate
group-transfer potential.
- ΔG of ATP hydrolysis varies with pH,
divalent metal ion (Mg2+) concentration,
and ionic strength.
- ATP hydrolysis under physiological
condition ΔGo = ~50 kJ/mol
- Under the biological standard condition,
ΔGo’ = -30.5 kJ/mol.
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Chapter 13
Takusagawa’s Note©
ΔG’s are additive, i.e., ΔG = ΣΔGi
-
Two examples are given in Fig. 15-21.
High energy bonds (~)
- produce ΔG < -25 kJ/mol by its hydrolysis.
- For example, ATP has two high energy bonds, ATP = AR-P~P~P.
- Why should the phosphoryl transfer reactions of ATP be so exergonic? Because
1. Poor resonance stabilization --- Resonance stabilization of a phosphoanhydride bond is less
than that of its hydrolysis products.
- Because π-electrons of bridging oxygen atom are drawn both phosphoryl groups (strong
competition), whereas there are no such competition in the hydrolyzed products.
O
O P
O
O
-
O
Poor resonance structure
P O
-
Repulsion
O
H2O
O
O P
O
O H
-
O
H O P O
-
O
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Good resonance structure
Takusagawa’s Note©
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Chapter 13
2. High electrostatic repulsion --- Destabilization effect of the electrostatic repulsions between
the charged groups of a phosphoanhydride. Under the physiological pH, ATP has 3~4
negative charges, indicating the presence of strong electrostatic repulsion in the molecule.
3. Small solvation energy --- Solvation energy of phosphoanhydride is smaller in comparison
to that of its hydrolysis products because all oxygen atoms cannot form H-bonds with water
molecules. The water shell of -P-P-P is quite poor (i.e., all oxygen atoms cannot form Hbonds with water molecules).
Other “High-energy” compounds
1. Acyl phosphates (Poor stabilization resonance and small solvation energy)
O
CH3
C
OH O
OPO3
2-
Acetyl phosphate
2-
C C OPO3
H
1,3-Bisphosphoglycerate
O3POCH 2
2-
2. Enol phosphates (After phosphoryl transfer, enol-keto tautomerization produces a relatively
large energy)
3. Phosphoguanidines (Competition of resonance in their guanidino group, i.e., poor resonance
form).
H3C
2-
NH2
N PO32H
Phosphocreatine
OOCH2C
N
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C
Takusagawa’s Note©
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Chapter 13
Low energy phosphate compounds
- Glucose-6-phosphate or glycerol-3-phosphate whose ΔG’s are below ATP.
- These molecules have less significantly different resonance stabilization or charge separation
in comparison with their hydrolysis products.
CH2OPO32CH2OH
O
HO
OH
OH
OH
H
CH2OPO32-
OH
α-D-glucose-6-phosphate
L-Glycerol-3-phosphate
C. Role of ATP
ATP occupies the middle rank of phosphoryl-transfer energy production
- This enables ATP to serve as an energy conduit between “high-energy” phosphate donors
and “low-energy” phosphate acceptors
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Chapter 13
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Takusagawa’s Note©
ATP is the universal energy currency of living system
- ATP is consumed by:
1. Early stage of nutrient breakdown (e.g., formation of glucose-6-phosphate and fructose-1,6bisphosphate).
2. Interconversion of nucleoside triphosphates.
ATP + NDP ↔ ADP + NTP (such as ATP + GDP ↔ ADP + GTP)
3. Physiological processes, such as:
- Muscle contraction.
- Transport of molecules and ions against concentration gradients (that requires
conformational changes in proteins/enzymes). e.g., (Na+-K+)-ATPase.
4. Reactions requires more than one ATP hydrolysis (e.g., 1st step of fatty acid oxidation).
- Additional phosphoanhydride cleavage is highly exergonic reactions.
- In normal process: ATP hydrolysis energy (ATP → ADP + Pi ΔG°’= -30.5 kJ/mol) is
enough to complete a reaction, but some processes are required more energy. If this is
the case, an ATP is degraded to AMP and 2Pi.
ΔG°’ = -32.2 kJ/mol)
ATP → AMP + PPi
PPi → 2Pi
ΔG°’ = -33.5 kJ/mol)
Total
=
ΔG°’ = -65.7 kJ/mol ← Maximum ATP hydrolysis energy
Formation of ATP
- ATP must be replenished.
1. Substrate-level phosphorylation.
e.g., Phosphoenolpyruvate + ADP → Pyruvate + ATP
2. Oxidative phosphorylation and photophosphorylation.
- Discharge of proton concentration gradient is enzymatically coupled to formation of ATP
from ADP.
3. Adenylate kinase reaction.
- AMP resulting from pyrophosphate cleavage reaction of ATP is converted to ADP by
adenylate kinase, and the ADP is converted to ATP by 1 or 2 processes.
AMP + ATP → 2ADP (then ADP → ATP)
Rate of ATP turnover
- Amount of ATP in a cell is typically only enough to supply for a minute or two.
- Brain cells have only a few seconds supply of ATP.
- Thus, rate of ATP turnover must be very fast.
- Phosphocreatine is a “high-energy” reservoir of ATP. The excess of ATP is stored as
phosphocreatine.
ATP + Creatine ↔ Phosphocreatine + ADP
ΔG°’ = 12.6 kJ/mol
- Under standard condition, this reaction is endergonic, however, the intercellular
concentration of reactants (high) and products (low) are such that its operates close to
equilibrium (ΔG ≈ 0).
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Chapter 13
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Takusagawa’s Note©
5. OXIDATION-REDUCTION REACTIONS
- In oxidation-reduction reactions, the groups transferred are electrons.
Electron donor (reductants or reducing agents)
↓
Electron acceptor (oxidants or oxidation agents)
- For example, Fe3+ + Cu+ ↔ Fe2+ + Cu2+
-
-
Half-reactions or redox couples:
Fe3+ + e- ↔ Fe2+
[reduction]
Cu+ ↔ Cu2+ + e[oxidation]
These half-reactions occur during oxidative metabolism in the vital mitochondrial electron
transfer mediated by cytochrome c oxidase.
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Takusagawa’s Note©
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Chapter 13
Electrochemical Cells
- Fe3+ and Fe2+ are a conjugate redox pair in the previous example.
Aoxn+ + Bred ↔ Ared + Boxn+ ------ (Aoxn+ and Ared) and (Bred and Boxn+) are conjugate pairs.
- Free energy change is:
⎛ [ Ared ] Boxn+ ⎞
o
⎟
ΔG = ΔG + RT ln⎜⎜ n +
[15.2]
⎟
A
B
⎝ ox [ red ] ⎠
- Under reversible condition, ΔG = -w’ = -wel
[15.3]
- w’ is the non-pressure-volume work (P = constant and V = constant)
- wel is the electrical work that requires to transfer the n moles of electrons through the electric
potential difference ΔE.
wel = nFΔE
[15.4]
F is the Faraday constant, the electrical charge of one mol of electrons (1F = 96,494 C/mol =
1 C = 1 Coulomb = 1 J/V
96,494 J·V-1·mol-1).
- Thus, ΔG = -nFΔE
[15.5]
Put this relation into [15.2]
⎛ [ Ared ] Boxn+ ⎞
⎟
− nFΔE = − nFΔE o + RT ln⎜⎜ n +
⎟
A
B
[
]
⎝ ox
red ⎠
[ ]
[ ]
ΔE = ΔE
-
o
[ ]
[ ]
RT ⎛ [ A ][ B ]⎞
⎟
−
ln ⎜
nF ⎜⎝ [ A ][ B ] ⎟⎠
red
n+
ox
n+
ox
red
[15.6]
This is Nernst equation.
ΔE is called redox potential or electromotive force (emf).
ΔE° is the standard redox potential in the standard state.
Measurements of redox potentials
- Free energy change ΔG can be determined by simply measuring its redox potential (ΔE) with
volt meter and converting it to ΔG using equation [15.5].
- Let consider a conjugated redox pair.
Aoxn+ + ne- ↔ Ared
Boxn+ + ne- ↔ Bred
The redox potentials in each pair are:
RT ⎛ [ Ared ] ⎞
⎟
E A = E Ao −
ln ⎜
[15.7a]
nF ⎜⎝ Aoxn+ ⎟⎠
[ ]
RT ⎛ [ Bred ] ⎞
⎟
ln⎜
[15.7b]
nF ⎜⎝ Boxn+ ⎟⎠
For the redox reaction of any two half-reactions:
ΔE° = E°(e-acceptor) - E°(e-donor)
[15.8]
E B = E Bo −
-
-
[ ]
In the above example, if A is electron acceptor and B is electron donor, then
ΔE° = E°A - E°B
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Chapter 13
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Takusagawa’s Note©
Standard reduction potentials (E°)
- are defined with respect to the standard hydrogen half-reduction, i.e.,
2H+ + 2e- ↔ H2(gas) at pH 0 (1 mol of H+), 25°C and 1 atm with a Pt electrode. This
reduction potential is set to be E° = 0 V.
- The hydrogen half-cell at the Biological standard state (pH 7) has E°’ = -0.421 V.
Some biochemically important half-reactions are listed in Table 15-4.
⎫
⎥
⎥
⎬ Spontaneous
⎥ reaction
⎥
⎭
Note:
- Positive ΔE gives negative ΔG, i.e., a positive ΔE is indicative of a spontaneous reaction, i.e.,
ΔG = -nFΔE.
- The more positive the standard reduction potential, the higher electron affinity of the redox
couple’s oxidized form. O2 has the strongest electron affinity in Table 15-4.
- O2 is the strongest oxidizing agent, whereas the weakest reducing agent.
-
Biochemical half-reactions are physiologically significant.
e.g., Fe3+ ions of the various cytochromes have significantly different redox potentials.
-
Electron-transfer reactions are of great biological importance.
e.g., electrons are passed from NADH along a series of chain reactions to O2. This free
energy cascade is used to generate ATP from ADP & Pi.
Concentration cells
17
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Takusagawa’s Note©
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Chapter 13
A concentration gradient requires the input of free energy of its formation.
Discharge of a proton concentration gradient drives the enzymatic synthesis of ATP from
ADP and Pi.
+
High [H ] or Low pH
+
H
Proton pump
Membrane
+
H
+
+
H
H
ADP + Pi
ATP
ATP synthetase
synthase
+
Low [H ] or High pH
ATP
-
Nerve impulses are transmitted through the discharge of [Na+] and [K+] gradients that nerve
cells generate across their cell membranes.
6.
-
THERMODYNAMICS OF LIFE
Living systems cannot be at equilibrium.
Living things maintain the steady state.
In a simple system such as:
A→B→C
If the system maintains the steady state, [B] = constant.
e.g., a man eats 10 lb of meat (A) per week and he digests the meat by 100%, but his weight
(B) stays a constant, because he uses various energy (C) in order to alive.
The steady state of an open system is therefore analogous to the equilibrium state of a closed
system, both are stable state.
-
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