Metabolism II and Glycolysis 5/7/03

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Metabolism II and Glycolysis 5/7/03
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Organic reaction mechanisms
Much can be learned by studying organic model
reactions when compared to enzyme catalyzed
reactions.
1. Group transfer reactions 2. Oxidations and
reductions 3. Eliminations, isomerizations and
rearrangements 4. Reactions that make or break
carbon-carbon bonds
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ATP ATP is the energy carrier for most biological
reactions
ATP H2O -gt ADP Pi ATP H2O -gt AMP PPi
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Coupled Reactions
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Recycling ATP ADP
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Heterolytic cleavage or bond formation is
catalyzed using either nucleophiles or
electrophiles.
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Nucleophiles
Basic reaction of amine
Nucleophilic reaction of an amine
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Biologically important nucleophiles
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Carbinolamine intermediate
Ketone or aldehyde
Amine
Imine
Movement of an electron pair from a position and
pointing to the electron deficient center
attracting the pair.
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Common biological electrophiles
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Group transfer reactions
Acetyl group transfer Nucleophile attack on an
acyl carbonyl to form a tetrahedral
intermediate Peptide bond hydrolysis Phosphoryl
group transfer nucleophile attack on a phosphate
to yield a trigonal bipyramid
intermediate Kinase reactions involving
transfer of phosphate from ATP to organic
alcohols Glycosyl group transfers substitution
of one group at the C1 carbon of a sugar for
another
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Thioesters (Acetyl-coenzyme A)
High energy compound Carrier of acetyl and acyl
groups Can be used to drive exogenic
processes e.g. GTP from GDP
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Oxidations and reductions
Oxidation Loss of Electrons Reduction Gain of
Electrons
Many redox reactions involve the breaking of a
C-H bond and the loss of two bonding electrons
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Electron transfer reactions to oxygen undergo
transfer of one electron at a time (Pauli
exclusion principle) Oxidations to oxygen from
NADH require two electron steps to be changed to
one electron steps. Stable radical structures
like FMN or FAD and cytochromes are involved.
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Reduction of NAD to NADH
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Electron transfer reactions
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Half-cell reactions either donate or accept
electrons Electron donor (reducing
agent) Electron acceptor (oxidizing agent)
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Nernst Equation- electromotive force -EMF-
reduction potential
Work is non -pressure volume work or DG -w
-welec Welec nFDE or DG -nFDE
F Faraday constant 96,485 Coulombs per mole
of electrons DE0 standard reduction potential
or midpoint potential
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Measuring potentials
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However, there is no absolute potential to
reference!
At equilibrium and in contact with a platinum
electrode and at 1 M H and STP this is defined
as zero potential. At pH of 7.0 this is
-0.421 V Eo. Prime means that it is at pH
7.0. Every thing is referenced to this potential
See Table in FOB pg 373 for standard potentials
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Metabolic pathways are irreversible
They have large negative free energy changes to
prevent them running at equilibrium. If two
pathways are interconvertible (from 1 to 2 or 2
to 1), the two pathways must be different!
Independent routes means independent control of
rates.
A
2
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The need to control the amounts of either 1 or 2
independent of each other.
X
Y
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Every pathway has a first committed step
A committed step is an irreversible step that
commits the pathway to the synthesis of the end
product. This step is usually the regulated step
in the pathway.
All metabolic pathways are regulated
The control of the flux through a pathway is
regulated by regulatory enzymes at the committed
step in the pathway. This control over
metabolism allows the organism to make
corrections and adjust to unforeseen changes.
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Pathways in eukaryotic cells occur in separate
organelles or cellular locations
ATP is made in the mitochondria and used in the
cytosol. Fatty acids are make in the cytosol and
broken down in the mitochondria. Separation of
pathways exerts a greater control over opposing
pathways and the intermediates can be controlled
by transport across the separating membranes.
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Experimental approaches to study metabolism
1. Sequence of reactions by which a nutrient is
converted to end products 2. Mechanism by which
an intermediate is turned into its successor. 3.
Regulation of the flow of metabolites in a
pathway. Inhibitors and growth studies are used
to see what is blocked. If a reaction pathway is
inhibited products before the block increase and
intermediates after the block decrease in
concentration
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Genetic Defects cause intermediates to accumulate
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Genetic manipulations can cause a block to occur
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Radioactive tracers
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Glycolysis

• The conversion of glucose to pyruvate to yield
  2ATP molecules
• 10 enzymatic steps
• Chemical interconversion steps
• Mechanisms of enzyme conversion and intermediates
• Energetics of conversions
• Mechanisms controlling the Flux of metabolites
  through the pathway

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Historical perspective

• Winemaking and baking industries
• 1854-1865 Louis Pasture established that
  microorganisms were responsible for fermentation.
• 1897 Eduard Buchner- cell free extracts carried
  out fermentation
• no vital force and put fermentation in the
  province of chemistry
• 1905 - 1910 Arthur Harden and William Young
• inorganic phosphate was required ie.
  fructose-1,6- bisphosphate
• zymase and cozymase fractions can be separated
  by diaylsis

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Inhibitors were used. Reagents are found that
inhibit the production of pathway products,
thereby causing the buildup of metabolites that
can be identified as pathway intermediates. Fluori
de- leads to the buildup of 3-phosphoglycerate
and 2-phosphoglycerate 1940 Gustav Embden, Otto
Meyerhof, and Jacob Parnas put the pathway
together.
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Pathway overview
1. Add phosphoryl groups to activate glucose. 2.
Convert the phosphorylated intermediates into
high energy phosphate compounds. 3. Couple the
transfer of the phosphate to ADP to form
ATP. Stage I A preparatory stage in which
glucose is phosphorylated and cleaved to yield
two molecules of glyceraldehyde-3-phosphate -
uses two ATPs Stage II glyceraldehyde-3-phosphate
is converted to pyruvate with the concomitant
generation of four ATPs-net profit is 2ATPs per
glucose. Glucose 2NAD 2ADP 2Pi ? 2NADH
2pyruvate 2ATP 2H2O 4H
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Oxidizing power of NAD must be recycled
NADH produced must be converted back to NAD
1. Under anaerobic conditions in muscle NADH
reduces pyruvate to lactate (homolactic
fermentation). 2. Under anaerobic conditions in
yeast, pyruvate is decarboxylated to yield CO2
and acetaldehyde and the latter is reduced by
NADH to ethanol and NAD is regenerated
(alcoholic fermentation). 3. Under aerobic
conditions, the mitochondrial oxidation of each
NADH to NAD yields three ATPs
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Hexokinase
Mg
ATP
ADP H
Glucose
Glucose-6-phosphate
Isozymes Enzymes that catalyze the same reaction
but are different in their kinetic
behavior Tissue specific Glucokinase- Liver
controls blood glucose levels. Hexokinase in
muscle - allosteric inhibition by ATP Hexokinase
in brain - NO allosteric inhibition by ATP
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Hexokinase reaction mechanism is RANDOM Bi-Bi
Glucose ATP ADP
Glu-6-PO4
When ATP binds to hexokinase without glucose it
does not hydrolyze ATP. WHY? The binding of
glucose elicits a structural change that puts the
enzyme in the correct position for hydrolysis of
ATP.
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The enzyme movement places the ATP in close
proximity to C6H2OH group of glucose and excludes
water from the active site.
There is a 40,000 fold increase in ATP hydrolysis
upon binding xylose which cannot be
phosphorylated!
a-D-Xylose
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Yeast hexokinase, two lobes are gray and green.
Binding of glucose (purple) causes a large
conformational change. A substrate induced
conformational change that prevents the unwanted
hydrolysis of ATP.
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Phosphoglucose Isomerase
Uses an ene dione intermediate 1) Substrate
binding 2) Acid attack by H2N-Lys opens the
ring 3) Base unprotonated Glu abstracts proton
from C2 4) Proton exchange 5) Ring closure
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Uncatalyzed isomerization of Glucose
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Phosphofructokinase
Mg
ATP
ADP
Fructose-6-PO4
Fructose-1,6-bisphosphate 1.) Rate limiting step
in glycolysis 2.) Irreversible step, can not go
the other way 3.) The control point for
glycolysis
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Aldolase
Dihydroxyacetone phosphate (DHAP)

Glyceraldehyde-3-phosphate (GAP)
Fructose -1,6-bisphosphate (FBP)
Aldol cleavage (retro aldol condensation)
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There are two classes of Aldolases
Class I animals and plants - Schiff base
intermediate Step 1 Substrate binding Step 2 FBP
carbonyl groups reacts with amino LYS to form
iminium cation (Schiff base) Step 3. C3-C4 bond
cleavage resulting enamine and release of
GAP Step 4 protonation of the enamine to a
iminium cation Step 5 Hydrolysis of iminium
cation to release DHAP
NaBH4
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Class II enzymes are found in fungi and algae and
do not form a Schiff base. A divalent cation
usually a Zn2 polarizes the carbonyl
intermediate.
Probably the occurrence of two classes is a
metabolic redundancy that many higher organisms
replaced with the better mechanism.
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Aldolase is very stereospecific
When condensing DHAP with GAP four possible
products can form depending on the whether the
pro-S or pro R hydrogen is removed on the C3 of
DHAP and whether the re or si face of GAP is
attacked.
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Triosephosphate isomerase
DHAP GAP
TIM is a perfect enzyme which its rate is
diffusion controlled. A rapid equilibrium allows
GAP to be used and DHAP to replace the used GAP.
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TIM has an enediol intermediate
GAP
enediol
DHAP
Transition state analogues Phosphoglycohydroxamate
(A) and 2-phosphoglycolate (B) bind to TIM 155
and 100 times stronger than GAP of DHAP
B.
A.
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TIM has an extended low barrier hydrogen bond
transition state
Hydrogen bonds have unusually strong interactions
and have lead to pK of Glu 165 to shift from 4.1
to 6.5 and the pK of
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Geometry of the eneolate intermediate prevents
formation of methyl glyoxal
Orbital symmetry prevents double bond formation
needed for methyl glyoxal