Transcript Enzyme Mechanisms - Illinois Institute of Technology

General Metabolism
Principles; Nutrition
Andy Howard
Introductory Biochemistry
1 December 2009
Biochemistry: Metabolism I
12/1/2009
Metabolism depends strongly
on cofactors
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We’ll attend to the reality that a lot of the
versatility of enzymes depends on their
incorporation of cofactors; and most
vitamins are precursors of cofactors
Biochemistry: Metabolism I
12/1/2009
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What we’ll discuss
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Post-translational
modification
Phosphorylation
Other reversible PTMs
How pathways evolve
Oxidation-Reduction
Reactions:
Quantitation
How we study
metabolism (revisited)
Biochemistry: Metabolism I
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Nutrition
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Macronutrients
Micronutrients
Specific cofactors
and vitamins
reconsidered
Fat soluble vitamins
Ascorbate
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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)
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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)
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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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Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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!
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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.
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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Macronutrients
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Proteins
Carbohydrates
Lipids
Fiber
Biochemistry: Metabolism I
12/1/2009
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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 aketoacids and incorporation into TCA)
All of the essential amino acids must be
supplied in adequate quantities
Biochemistry: Metabolism I
12/1/2009
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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)
Biochemistry: Metabolism I
12/1/2009
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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 I
AA
moles
ATP
Arg 44
Pro 39
Ser 18
Gly 12
Cys 19
Phe 65
Tyr 62
Trp 78
His 42
12/1/2009
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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
Biochemistry: Metabolism I
12/1/2009
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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
arachidonate aren’t considered vitamins
Biochemistry: Metabolism I
12/1/2009
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Lipid vitamins
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Contain rings & long aliphatic
sidechains
At least one polar group in
each
Absorbed in intestine, carried
via bile salts
Hard to study
Most are formally built from
isoprene units, as are steroids
Biochemistry: Metabolism I
12/1/2009
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Vitamin A (retinol)
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3 forms varying in terminal polar group
Involved in signaling and receptors
b-carotene is nonpolar dimer
Biochemistry: Metabolism I
12/1/2009
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Vitamin A deficiency
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Produces night blindness because the
retina and cornea dry out
Most common cause: nursing infants
whose mothers have vitamin A deficiency
in their diet
Biochemistry: Metabolism I
12/1/2009
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Vitamin D
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Several related forms
Hormones involved in
Ca2+ regulation
Figure courtesy
Cyberlipid
Biochemistry: Metabolism I
(cholecalciferol)
12/1/2009
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Vitamin D deficiency
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Rickets in children:
Bone disease, restlessness, slow growth
One form of vitamin D is actually
synthesizable from cholesterol given
adequate sunlight;
Therefore rickets is most common in
densely settled urban environments
Biochemistry: Metabolism I
12/1/2009
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Vitamin E (a-tocopherol)
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Phenol can undergo 1e- oxidation to
moderately stable free radical
Antioxidant activity prevents damage to
fatty acids in membranes
phenol
Fig. Courtesy
UIC pharmacy program
Biochemistry: Metabolism I
12/1/2009
Page 43 of 50
Vitamin K (phylloquinone)
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Involved in synthesis of
proteins involved in
blood coagulation
Reduced form involved
as reducing agent in
carboxylation reaction
on glu sidechains
Figure courtesy
Cyberlipid
Biochemistry: Metabolism I
12/1/2009
Page 44 of 50
Vitamin
overdoses?
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
It’s difficult to overdose on water-soluble
vitamins: excess is simply excreted
Fat-soluble vitamins are stored in adipose
tissue and can accumulate to high
concentrations
May be toxic even dietarily
Therefore: don’t eat polar bear liver
Biochemistry: Metabolism I
12/1/2009
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Ascorbate
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The only common water-soluble vitamin that
is not a coenzyme or coenzyme precursor
Vitamin in primates, some rodents
Synthesizable in most other vertebrates
Involved in collagen
Reduced form acts as reducing agent during
hydroxylation of collagen
Deficiency gives rise to inadequate collagen scurvy
Biochemistry: Metabolism I
12/1/2009
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PTM role of ascorbate
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Proline + O2 + a-ketoglutarate + ascorbate 
4-hydroxyproline + succinate + CO2 +
dehydroascorbate
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This is a post-translational modification that
occurs to prolines within collagen
The hydroxylated prolines help stabilize the
collagen triple helix
Hydroxylysine found in collagen too
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Biochemistry: Metabolism I
12/1/2009
Page 47 of 50
Dietary deficiency
of ascorbate
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Primary sources of
ascorbate are fruits,
particularly citrus, and
green vegetables
Ascorbate deficiency’s
first symptom involves
collagen degradation,
leading to scurvy
Biochemistry: Metabolism I
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Image courtesy
U.Cincinnati
Medical School
12/1/2009
Page 48 of 50
Scurvy in history
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Shortage of green vegetables in
sailors’ diets meant scurvy was
rampant on shipboard until the
18th century
Success of English navy over
French 1760-1800 was partly due
to the introduction of limes in
English sailors’ diets 50 years
before the French caught on
Biochemistry: Metabolism I
12/1/2009
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Page 49 of 50
Megadoses of
ascorbate
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Linus Pauling (2-time Nobel
laureate) became convinced late
in his life that very high doses of
ascorbate (> 1 g /day) were
beneficial as a preventative
His assertions were met with
skepticism from the established
medical community
I would say the jury is still out!
Biochemistry: Metabolism I
12/1/2009
Linus Pauling
Image courtesy
Oregon State U.
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