Transcript General Metabolism II - Illinois Institute of Technology

General Metabolism II
Andy Howard
Introductory Biochemistry, fall 2010
18 November 2010
Biochemistry: Metabolism II
11/18/2010
Metabolism:
the core of biochem
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All of biology 402 will concern itself with
the specific pathways of metabolism
Our purpose here is to arm you with the
necessary weaponry
… but first, we need to explain the role of
Ca2+ in muscle contraction
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What we’ll discuss
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Metabolism
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Control
Feedback
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Flux
Phosphorylation
Other PTMs
Evolution
Redox
Tools for studying
Biochemistry: Metabolism II
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Nutrition
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Macronutrients
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Proteins
Fats
Carbohydrates
Vitamins
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Fat-soluble
Water-soluble
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iClicker quiz question 1
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An asymmetry between stage 1 of catabolism
(C1) and the final stage of anabolism (A3) is
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(a) A3 always requires light energy; C1 doesn’t
(b) A3 never produces nucleotides;
C1 can involve nucleotide breakdown
(c) A3 adds one building block at a time to the
end of the growing polymer;
C1 can involve hydrolysis in the middle of the
polymer
(d) There are no asymmetries between A3 and
C1
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iClicker quiz question 2
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Could dAMP, derived from degradation of
DNA, serve as a building block to make
NADP?
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(a) Yes.
(b) Probably not: the energetics wouldn’t
allow it.
(c) Probably not: the missing 2’-OH would
make it difficult to build NADP
(d) No: dAMP is never present in the cell
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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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Metabolic flux, illustrated
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Courtesy Jeremy Zucker’s wiki
http://bio.freelogy.org/wiki/User:JeremyZucker#Metabolic_Engineering_tutorial
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Feedback and
Feed-forward
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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)
 Controlling element is usually the
last element in the path
 Often the controlled reaction has a
large negative Go’.
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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
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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
Biochemistry: Metabolism II
Muscle phosphorylase
EC 2.4.1.1
192kDa dimer
monomer shown
PDB 2GJ4, 1.6Å
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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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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
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Are there other reversible posttranslational modifications 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
Cis-trans isomerization of
prolines
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ADP-ribosylation
of arginine; fig.
courtesy RPI
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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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Oxidation-reduction
reactions and Energy
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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
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This can be expressed thus:
Go’ = -nFEo’
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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]
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This is the Nernst equation
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Free energy from
electron transfer
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We can examine tables of
electrochemical half-reactions to get an
idea of the yield or requirement for
energy in redox reactions
Example:
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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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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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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Nutrition
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Lots of nonsense,
some sense on this subject
Skepticism among MDs as to its
relevance
Fair view is that nutrition matters in
many conditions, but it’s not the only
determinant of health
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Macronutrients
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Proteins
Carbohydrates
Lipids
Fiber
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Protein as food
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Source of essential amino acids
Source of non-essential aa
Fuel (often via interconversion to ketoacids and incorporation into TCA)
All of the essential amino acids must be
supplied in adequate quantities
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Which amino acids are
essential?
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At one level, that’s an easy question to answer:
they’re the ones for which we lack a biosynthetic
pathway: KMTVLIFWH
That shifts the question to:
why have some of those pathways survived and
not all?
Answer: pathways that are complex or require
more than ~30 ATP / aa are absent (except R,Y)
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The human list
AA
Asp
Asn
Lys
Met
Thr
Ala
Val
Leu
Ile
moles
ATP
21
22-24
50-51
44
31
20
39
47
55
essential?
no
no
yes
yes
yes
no
yes
yes
yes
Glu
Gln
30
31
no
no
Biochemistry: Metabolism II
AA
moles
ATP
Arg 44
Pro 39
Ser 18
Gly 12
Cys 19
Phe 65
Tyr 62
Trp 78
His 42
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essential?
no
no
no
no
no
yes
no*
yes
yes
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Carbohydrates as food
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Generally recommended to be more than
half of caloric intake
Complex carbohydrates are hydrolyzed
to glucose-1-P and stored as glycogen or
interconverted into other metabolites
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Lipids as food
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You’ll see in 402 that the energy content
of a lipid is ~ 2x that of carbohydrates
simply because they’re more reduced
They’re also more efficient food storage
entities than carbs because they don’t
require as much water around them
Certain fatty acids are not synthesizable;
by convention we don’t call those
vitamins
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Vitamins
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Vitamins are necessary micronutrients
A molecule that is a vitamin in one organism
isn’t necessarily a vitamin in another
E.coli can make all necessary metabolites
given sources of water, nitrogen, and carbon
Most eukaryotic chemoautotrophs find it more
efficient to rely on diet to make complex
metabolites
We’ll discuss lipid vitamins first,
then water-soluble vitamins
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Why wouldn’t organisms
make everything?
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Complex metabolites require energy for
synthesis
Control of their synthesis is also
metabolically expensive
Cheaper in the long run to derive these
nutrients from diet
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Vitamins: broad classifications
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Water-soluble vitamins
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Coenzymes or coenzyme precursors
Non-coenzymic metabolites
Fat-soluble vitamins
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Antioxidants
Other lipidic vitamins
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Are all nutrients that we can’t
synthesize considered
vitamins?
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No:
If it’s required in large quantities,
it’s not a vitamin
By convention, essential fatty acids like
linoleate aren’t considered vitamins
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