Metabolism
Fundamentals
Andy Howard
Introductory Biochemistry, Spring 2009
11 March 2009
Biochemistry: metabolism
1
03/11/2009
Metabolic principles
learned here. . .
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… will be useful throughout the
remainder of the course
We’ll need concepts of energy flux,
feedback, feed-forward, posttranslational modification,
thermodynamics, kinetics, reduction
potential, . . .
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What we’ll discuss

Metabolism
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Definitions
Pathways
Control
Feedback
Phosphorylation
Thermodynamics
Kinetics
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ATP
Thioesters
Oxidation-reduction
reactions
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Potential
Energetics
NAD(P)
How we study
metabolism
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Metabolism
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Almost ready to start the specifics
(chapter 11)
Define it!
Metabolism is the network of chemical
reactions that occur in biological
systems, including the ways in which
they are controlled.
So it covers most of what we do here!
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Intermediary Metabolism
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Metabolism involving small molecules
Describing it this way is a matter of
perspective:
Do the small molecules exist to give the
proteins something to do, or do the
proteins exist to get the metabolites
interconverted?
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Metabolic pathways
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We can understand metabolic pathways
in terms of macromolecular behavior as
well as small-molecule behavior.
Cofactors and vitamins are components
of those pathways
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Pathway
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A sequence of reactions such that
the product of one is the substrate
for the next
Similar to an organic synthesis
scheme
(but with better yields!)
May be:
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Unbranched
Branched
Circular
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Metabolic pathways
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Anabolism: buildup of complex molecules
from simple ones, generally with the
insertion of energy in the form of ATP
hydrolysis
Catabolism: breakdown of complex
molecules into simpler ones, usually with
release of energy in the form of ATP
production or reduction of NAD to NADH
Amphibolism: Overlap of anabolism with
catabolism within one pathway
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Why multistep pathways?
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Limited reaction specificity of
enzymes
Control of energy input and output:
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Break big inputs into ATP-sized inputs
Break energy output into pieces that
can be readily used elsewhere
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Anabolic Pathways
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Buildup of complex molecules
Specific pathways:
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Gluconeogenesis (ch.12)
TCA cycle and glyoxalate pathway (12-13)
Calvin cycle (chapter 15)
Starch and glycogen synthesis (12)
Nucleotide and amino acid synthesis
(chapters 17,18)
Fatty acid and lipid synthesis (chapter 16)
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Anabolic pathways

Horton 10.5; this from Citizendium.org
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Anabolic divergence
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A few simple precursor molecules get
combined and modified to form many
end products
Building blocks generated from various
metabolites, e.g.:
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-amino acids derived by (trans)amination
of -ketoacids
Fatty acids built up two carbons at a time
from acetyl CoA
Carbohydrates built up from pyruvate
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Catabolic pathways
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Energy-yielding oxidations
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Breakdown of storage molecules
(chapter 12)
Breakdown of N-containing molecules
(chapter 17)
Glucose to TCA cycle (chapters 11,13)
TCA cycle (chapter 13)
Electron transport and oxidative
phosphorylation (chapter 14)
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Catabolic pathways

Horton 10.6; this from citizendium.org
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Catabolism: convergence
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Stage 1: break nutrients into
building blocks
Stage 2: break building blocks
into a very small number of end
products
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Note that some pathways are
both anabolic and catabolic!
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These pathways are amphibolic
Specific metabolites are both intermediaries in
these pathways and they’re useful in other
contexts
If a metabolite is depleted out of a pathway,
we generally need a replenishment reaction to
rebalance things
Replenishment reactions are called anapleurotic
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Are build-up and breakdown
identical?
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No
Energetics say we can’t do that
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Some enzymes (catalyzing nearly
isoergic reactions) are shared
Others differ in ATP or other energy
requirements
Control elements can be different too
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Common metabolic themes
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Maintenance of internal concentrations
of ions, metabolites, enzymes
Extraction of energy from external
sources
Pathways specified genetically
Organisms & cells interact with their
environment
Constant degradation & synthesis of
metabolites and macromolecules to
produce steady state
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Metabolism and energy
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Energy & carbon
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Autotrophs
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QuickTime™ and a
decompressor
are needed to see this picture.
Photoautotrophs:
get energy from light and C from CO2 Methanopyrus,
a chemiautotroph
Chemiautotrophs (bacterial only)
(Wikipedia)
get energy from food and C from CO2
Heterotrophs
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Photoheterotrophs (bacterial only):
energy from light,
require organic carbon
Chemoheterotrophs:
energy from food,
require organic carbon
03/11/2009 Biochemistry: metabolism
QuickTime™ and a
decompressor
are needed to see this picture.
Theocapsa,
A photoheterotroph
Kenyon microwiki
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Energy flow
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Energy flows from the sun via
photosynthesis and then flows through
biological systems through the ingestion
of food, generation of heat, and other
thermodynamic processes
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The role of oxygen
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Oxygen is necessary to survival in most
organisms—namely, aerobic organisms—where
it functions as the final electron acceptor for the
electron transport chain.
It is, however, toxic because it’s reactive in ways
that are often deleterious.
Many mechanisms exist for detoxifying the
undesirable side-products of oxygen
metabolism, particularly in aerobic organisms,
where the organism can’t simply escape O2.
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Regulation
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Organisms respond to change
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Fastest: small ions move in msec
Metabolites: 0.1-5 sec
Enzymes: minutes to days
Flow of metabolites is flux:
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steady state is like a leaky bucket
Addition of new material replaces the
material that leaks out the bottom
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Feedback and
Feed-forward

Mechanisms by
which the
concentration of a
metabolite that is
involved in one
reaction influences
the rate of some
other reaction in the
same pathway
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Feedback realities
Control usually exerted at first
committed step (i.e., the first
reaction that is unique to the
pathway)
 Otherwise, it occurs on irreversible
steps
 Controlling element is usually the
last element in the path

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Feed-forward
Early metabolite activates a reaction
farther down the pathway
 Has the potential for instabilities,
just as in electrical feed-forward
 Usually modulated by feedback
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Activation and inactivation by
post-translational modification
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Most common:
covalent phosphorylation of protein
usually S, T, Y, sometimes H
Kinases add phosphate
Protein-OH + ATP 
Protein-O-P + ADP
… ATP is source of energy and Pi
Phosphatases hydrolyze phosphoester:
Protein-O-P +H2O Protein-OH + Pi
… no external energy source required
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Phosphorylation’s effects
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Phosphorylation of an enzyme can either
activate it or deactivate it
Usually catabolic enzymes are activated
by phosphorylation and anabolic enzymes
are inactivated
Example:
glycogen phosphorylase is activated by
phosphorylation; it’s a catabolic enzyme
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Glycogen phosphorylase
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Reaction: extracts 1 glucose
unit from non-reducing end of
glycogen & phosphorylates it:
(glycogen)n + Pi 
(glycogen)n-1 + glucose-1-P
Activated by phosphorylation
via phosphorylase kinase
Deactivated by
dephosphorylation by
phosphorylase phosphatase
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Amplification
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Activation of a single molecule of a
protein kinase can enable the
activation (or inactivation) of many
molecules per sec of target proteins
Thus a single activation event at the
kinase level can trigger many events
at the target level
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Other PTMs (G&G p. 505)
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Are there other reversible PTMs that
regulate enzyme activity? Yes:
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Adenylation of Y
ADP-ribosylation of R
Uridylylation of Y
Oxidation of cysteine pairs to cystine
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Metabolism and evolution
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Metabolic pathways have evolved over
hundreds of millions of years to work
efficiently and with appropriate controls
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Evolution of Pathways:
How have new pathways evolved?
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Add a step to an existing pathway
Evolve a branch on an existing pathway
Backward evolution
Duplication of existing pathway to create
related reactions
Reversing an entire pathway
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Adding a step
E1
E2
E3
E4
E5
ABCDEP
Original pathway
• When the organism makes lots of E,
there’s good reason to evolve an
enzyme E5 to make P from E.
• This is how asn and gln pathways
(from asp & glu) work
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Evolving a branch
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Original pathway:
D
E1 E2
A  B  C E3
X
Fully evolved pathway:
E3a D
ABC
E3b X
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Backward evolution
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Original system has lots of E  P
E gets depleted over time;
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Then D gets depleted;
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need to make it from D,
so we evolve enzyme E4 to do that.
need to make it from C,
so we evolve E3 to do that
And so on
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Duplicated pathways
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Homologous enzymes catalyze related
reactions;
this is how trp and his biosynthesis
enzymes seem to have evolved
Variant: recruit some enzymes from
another pathway without duplicating the
whole thing (example: ubiquitination)
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Reversing a pathway
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We’d like to think that lots of pathways are fully
reversible
Usually at least one step in any pathway is
irreversible (Go’ < -15 kJ mol-1)
Say CD is irreversible so E3 only works in the
forward direction
Then D + ATP C + ADP + Pi allows us to
reverse that one step with help
The other steps can be in common
This is how glycolysis evolved from
gluconeogenesis
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Enzyme organization
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Enzymatic reactions are organized into
pathways, where reactions proceed in an ordered
sequence leading from the first reactant to the
final product
Often, especially in eukaryotes, the relevant
enzymes are spatially organized into groupings
that allow one enzyme to emit its product in a
position where it can be immediately picked up
as a substrate by the next enzyme (G&G fig.
17.5)
These grouped enzymes are often membranebound to provide physical stability
Metabolons are stable multienzyme complexes
that work this way
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Compartmentation I:
Localized pathways
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Some are in membranes, some free
in cytosol or in aqueous organelles
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Several catabolic pathways in eukaryotes
are localized in mitochondria
Corresponding anabolic pathways are in
cytosol
Reduces likelihood of futile cycling
Multienzyme complexes, especially in
eukaryotes
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Provide entropic advantage
Often membrane-associated
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Compartmentation II:
Tissue Specialization
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Obvious in multicellular eukaryotes
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Individual cells may perform a limited number
of metabolic roles
Some fully mature cells are anuclear
Requires careful cell-cell communication
Even in cyanobacteria
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Thermodynamics
We did this carefully earlier:
this is just a reminder
 Remember that G is not Go’:

G = Go’ + RT ln[products]/[reactants]

At equilibrium G = 0,
so we can use that equation to find
Go’
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Practical biochemical
thermodynamics
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Most reactions are considered either
irreversible or fully reversible
Irreversible means Go’ < -20 kJ/mol;
Even with substrate and product
concentrations considered, the reaction
proceeds in only one direction
Reversible: -15 < Go’ < 15 but
G very close to zero
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N
ATP
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O
P
P
P
OH
OH
O
O
O-
O-
H2N
N
O
O
N
N

O
O
O-
Mg+2
HO
adenosine triphosphate
Both the anhydride bonds are considered
high-energy bonds; the phosphoester
bond is not.
Remember the story about
pyrophosphate!:
PPi + H2O  2Pi, Go’ = -29 kJ mol-1
Rapidity of this hydrolysis drives reactions
involving pyrophosphate to right
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ATP chemistry
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Why is ATP a high-energy
compound?
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Negative charges repel!
(somewhat mitigated by Mg2+)
ADP and Pi or AMP and PPi are better
solvated than ATP
More delocalization in products
Therefore ATP (and CTP, GTP, …)
are high-energy compounds
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Interconversions among
nucleotide phosphates
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Kinases or phospotransferases involved
Nucleoside monophosphate kinases:
ATP + XMP  ADP + XDP
specific to each X (G or dG, C or dC, …)
Nucleoside diphosphate kinase:
ATP + XDP  ADP + XTP
this is a single enzyme (why?)
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AnP interconversions

Adenylate kinase (special case of NMK…)
AMP + ATP  2ADP
 [ATP] >> [ADP] and [ATP] >> [AMP], so small
changes in [ATP] can drive big changes in
others:
[ATP],mM [ADP],mM [AMP],mM G1,kJmol-1
4.8
0.2
0.004
-40
4.5
0.5
0.02
-37
3.9
1.0
0.11
-35
3.2
1.5
0.31
-34

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How coupling works
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We tend to hand-wave about this
Enzymes provide location for
intermediates
X + ATP  X—P + ADP
X—P + Y + H2O  X-Y + Pi + H+
The X here can be an enzyme sidechain or a substrate
In the former case some other event
must come along to recreate X;
otherwise, it isn’t an enzyme!
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O
Glutamine
synthetase
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O
O
OP
-O
O
NH3+
gamma-glutamyl phosphate
Cf. section 25.2 - 25.3:
 glu + ATP  -glutamylphosphate + ADP
 -glutamylphosphate + NH3 -> gln + Pi
Why do we need ATP at all for this?
 Go’ = 14 kJ mol-1 for glu + NH3 -> gln + H2O
 we could overcome that with concentrations
 But we can’t: we need [glu] ~ [gln]
 So we need the energy charge
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O-
Go’hyd for metabolites
Transfers are more common than hydrolysis;
these help with bookkeeping
Go’hyd,
kJ mol-1
PEP
-62
1,3-bisPglycerate -49
ATP->AMP+PPi -45
Phosphocreatine -43
P-arginine
-32
Compound
Compound
Go’hyd
Acetyl CoA*
-32
ATP
-32
Pyrophosphate
-29
Glucose 1-P
-21
Glucose 6-P
-14
Glycerol 3-P
-9
* not a phosphate cmpd!
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HN
Making ATP
by transfer
NH2
O
P
O
N
-O
OH
O-
phosphocreatine
We’ve just seen that some compounds
have higher-energy phosphates than ATP
 Remember the 35-cent analogy
 So a phosphoryl-group transfer into ATP
can be energetically favorable, e.g.
Phosphocreatine + ADP  creatine + ATP

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Relative energies of
phosphate compounds
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Both phosphoenolpyruvate
(~ 60 kJ mol-1) and phosphocreatine
(~ 45 kJ mol-1) are higher-energy than
ATP (*what does that mean?)
ATP is therefore intermediate between
these high-energy phosphates and the
low-energy phosphates like glucose-6phosphate and glucose-1-phosphate
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ATP and the energy cycle
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Catabolism usually gives rise to energy
that is captured in high-energy
phosphate bonds in ATP
This ATP is used to provide energy for
otherwise endergonic reactions and to
phosphorylate things that need to be
phosphorylated
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Nucleotidyl-group transfer
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It’s most convenient to think of this as a
transfer of the entire nucleotide group to
form an acyl-adenylate intermediate
This can then fall apart, releasing AMP
and allowing a high-energy bond to form
Example: acetyl CoA (see pp.561-562)
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Thioesters: another class of
high-energy compounds

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Thioesters have similar reactiviy as
oxygen-acid anhydrides
Thioesters less stable than oxygen esters
because the unshared electrons in sulfur
are not as delocalized in a thioester as
the unshared electrons in an oxygen
ester
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Oxidation-reduction
reactions and Energy


Oxidation-reduction reactions involve
transfer of electrons, often along with
other things
Generally compounds with many C-H
bonds are high in energy because the
carbons can be oxidized (can lose
electrons)
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Reduction potential

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
Reduction potential is a measure of
thermodynamic activity in the context of
movement of electrons
Described in terms of half-reactions
Each half-reaction has an electrical
potential, measured in volts, associated
with it because we can (in principle)
measure it in an electrochemical cell
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So what is voltage, anyway?
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Electrical potential is available energy per
unit charge:
1 volt = 1 Joule per coulomb
1 coulomb = 6.24*1018 electrons
Therefore energy is equal to the potential
multiplied by the number of electrons
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Electrical potential and energy

This can be expressed thus:
Go’ = -nFEo’

n is the number of electrons transferred
F = fancy way of writing # of Coulombs
(which is how we measure charge) in a
mole (which is how we calibrate our
energies) = 96.48 kJ V-1mol-1

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Oh yeah?
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Yes.
1 mole of electrons = 6.022 * 1023 e1 coulomb = 6.24*1018 e1 mole = 9.648*104 Coulomb
1 V = 1 J / Coulomb=10-3 kJ / Coulomb
Therefore the energy per mole
associated with one volt is
10-3 kJ / C * 9.648*104 C = 96.48 kJ
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What can we do with that?


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The relevant voltage is the difference in
standard reduction potential between
two half-reactions
Eo’ = Eo’acceptor - Eo’donor
Combined with free energy calc, we see
Eo’ = (RT/nF ) lnKeq and
E = Eo’ - (RT/nF ) ln [products]/[reactants]

This is the Nernst equation
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Free energy from
electron transfer



We can examine tables of
electrochemical half-reactions to get an
idea of the yield or requirement for
energy in redox reactions
Example (see section 10.9B):
NADH + (1/2)O2 + H+ -> NAD+ + H2O;
We can break that up into half-reactions
to determine the energies
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Half-reactions and energy


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NAD+ + 2H+ + 2e-  NADH + H+,
Eo’ = -0.32V
(1/2)O2 + 2H+ + 2e-  H2O, Eo’ = 0.82V
Reverse the first reaction and add:
NADH + (1/2)O2 + H+  NAD+ + H2O,
Eo’ = 0.82+0.32V = 1.14 V.
Go’ = -nFEo’
= -2*(96.48 kJ V-1mol-1)(1.14V)
= -220 kJ mol-1; that’s a lot!
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NAD: electron collector



Net reactions involve transfer of hydride
(H:-) ions
Enzymes called dehydrogenases (a type
of oxidoreductase) involved
Collected NADH can then be reoxidized
in oxidative phosphorylation to drive ATP
synthesis in the mitochondrion
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NADPH


Provides reducing power for anabolic
reactions
Often converting highly oxidized
sugar precursors into less reduced
molecules
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



Absorbance
How to detect
NAD reactions
NAD+
340 nm
NADH
NAD+ and NADH
(and NADP+ and NADPH)
Wavelength
have extended aromatic systems
But the nicotinamide ring absorbs strongly
at 340 only in the reduced
(NADH, NADPH) forms
Spectrum is almost pH-independent, too!
So we can monitor NAD and NADPdependent reactions by appearance or
disappearance of absorption at 340 nm
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How much ATP can we get
out of oxidizing NADH?



In principle -220 kJ mol-1 should be
enough to drive production of at least five
ATP molecules
(220/32) = 6.9; even if we figure it will
cost more like 40 kJ mol-1 per ATP, then
that’s (220/40) = 5.5.
But in fact we only get about 3.5.
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Why?

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Short answer: discrete inefficiency
4 oxidation steps in the electron transport
chain beginning with NADH
3 of 4 of those steps facilitate transfer of
protons against a pH and charge gradient
When those protons move back across
with their charge and concentration
gradients, we earn ATP back
… but only about net 3.5 ATP per NADH
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Pathway methods
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Introducing inhibitors
Site-directed mutagenesis
Radioisotope tracing
Non-radioactive isotope tracing
NMR
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Classical metabolism studies
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Add substrate to a prep and look for
intermediates and end products
If substrate is radiolabeled (3H, 14C) it’s
easier, but even nonradioactive isotopes can
be used for mass spectrometry and NMR
NMR on protons, 13C, 15N, 31P
Reproduce reactions using isolated
substrates and enzymes
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Next level of sophistication…
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Look at metabolite concentrations in
intact cell or organism under relevant
physiological conditions
Note that Km is often ~ [S].
If that isn’t true, maybe you’re looking at
the non-physiological substrate!
Think about what’s really present in the
cell.
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Mutations in single genes
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If we observe or create a mutation in a
single gene of an organism, we can find
out what the effects on viability and
metabolism are
In humans we can observe genetic
diseases and tease out the defective
gene and its protein or tRNA product
Sometimes there are compensating
enzyme systems that take over when one
enzyme is dead or operating incorrectly
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Deliberate manipulations
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Bacteria and yeast:
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Irradiation or exposure to chemical mutagens
Site-directed mutagenesis
Higher organisms:
We can delete or nullify some genes;
thus knockout mice
Introduce inhibitors to pathways and see
what accumulates and what fails to be
synthesized
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