Transcript Lecture 20

Metabolism II and Glycolysis
5/7/03
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
ATP
ATP is the energy carrier for most biological
reactions
ATP + H2O -> ADP + Pi
ATP + H2O -> AMP + PPi
Coupled Reactions
Recycling ATP & ADP
Heterolytic cleavage or bond formation is catalyzed using either
nucleophiles or electrophiles.
Nucleophiles
H
Basic reaction
of amine
R
NH 2 + H+
R
N+
H
H
H
R'
N
C
R'
Nucleophilic reaction
of an amine
R
NH 2
O
R
R''
R''
OH
Biologically important nucleophiles
Carbinolamine
intermediate
Amine Ketone or aldehyde
R'
R'
R
NH 2
R
O
R''
N
C
H
R''
OH
Imine
R''
R
+
N
H
R'
Movement of an electron pair from a position and pointing to the
electron deficient center attracting the pair.
Common biological electrophiles
Group transfer reactions
Y
+
A
X
Y
A + X
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
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
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
H
R
Y
+
H
O
C
O
NH 2
H
+
N
R
R
H
H
R
Y
H
+
NH 2
+
O
O
R
R
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.
Reduction of NAD+ to NADH
Electron transfer reactions
A
n
ox
n
 Bred  Ared  Box
 A red   B 
G  G  RT ln  n   

 A ox   Bred 
0
n
ox
Half-cell reactions either donate or accept electrons
Electron donor (reducing agent)
Electron acceptor (oxidizing agent)
Nernst Equation- electromotive force -EMF- reduction potential
Work is non -pressure volume work or
G = -w’ = -welec
Welec = nFE
or
G = -nFE
 
RT  A red  B 

E  E ln  n 
nF  A ox Bred  
o
 
n
ox
F = Faraday constant = 96,485 Coulombs per mole of electrons
E0 = standard reduction potential or midpoint potential
Measuring potentials
A
n
ox
 ne  A red
-
and
B
n
ox
 ne  Bred
-

A red 
RT
EA  E Ln n 
nF
A ox
 
0
A
E  E
o
o
e- acceptor 
-E
o
e- donor
However, there is no absolute potential to reference!

2H  2e
-
 H 2 (g)
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
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
A
rates.
2
1
Y
X
The need to control the
amounts of either 1 or 2
independent of each other.
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.
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.
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
Genetic Defects cause intermediates
to accumulate
Genetic manipulations can cause a
block to occur
Radioactive tracers
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
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-1,6bisphosphate
•
zymase and cozymase fractions can be separated by
diaylsis
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.
Fluoride- leads to the buildup of 3-phosphoglycerate
and 2-phosphoglycerate
1940 Gustav Embden, Otto Meyerhof, and Jacob
Parnas put the pathway together.
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-3phosphate - 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+
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
Hexokinase
CH2OH
O
H
H
OH
Mg++
H
OH
H
OH
Glucose
O
H
H
OH
+ ATP
H
OH
CH2OPO32H
+ ADP + H+
H
OH
OH
H
OH
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
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.
The enzyme movement places the ATP in close
proximity to C6H2OH group of glucose and excludes
water from the active site.
H
O
H
H
OH
OH
H
OH
a-D-Xylose
There is a 40,000 fold
increase in ATP hydrolysis
upon binding xylose which
cannot be phosphorylated!
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.
Phosphoglucose Isomerase
CH2OPO32O
H
H
OH
H
O3POCH2
H
OH
OH
CH2OH
O
H
OH
H
-2
HO
H
OH
OH
H
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
Uncatalyzed isomerization of Glucose
Phosphofructokinase
-2
-2
O3POCH2
CH2OH
O
H
HO
H
+ ATP
OH
OH
H
Fructose-6-PO4
O3POCH2
CH2OPO3-2
O
Mg++
H
HO
H
+ ADP
OH
OH
H
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
Aldolase
CH2OPO3-2
CH2OPO3-2
C
O
HO
C
H
H
C
OH
H
C
OH
HO
C
O
C
H
H
+
O
H
CH2OPO3-2
H
Fructose -1,6-bisphosphate
(FBP)
Dihydroxyacetone
phosphate (DHAP)
C
OH
Glyceraldehyde-3phosphate (GAP)
CH2OPO3-2
Aldol cleavage (retro aldol condensation)
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
CH2OPO3-2
C14
CH2OH
NH
CH2OPO3-2
(CH2)4
Lys
+ NaBH4
H
C14
CH2OH
NH 3
(CH2)4
Lys
Class II enzymes are found in fungi and algae and
do not form a Schiff base. A divalent cation usually
a Zn+2 polarizes the carbonyl intermediate.
CH2OPO32-
CH2OPO3-2
C
HO
C
O
-
-
O
C
Zn 2+
HO
Zn 2+
H
H
Probably the occurrence of two classes is a metabolic
redundancy that many higher organisms replaced
with the better mechanism.
Aldolase is very stereospecific
When condensing DHAP with GAP four possible
products can form depending on the whether the proS or pro R hydrogen is removed on the C3 of DHAP
and whether the re or si face of GAP is attacked.
CH2OPO3
H
H
H
OH
OH
CH2OPO3
D-Fructose
1,6 bisphosphate
2-
CH2OPO32-
O
O
O
HO
CH2OPO32-
CH2OPO32-
2-
O
H
OH
HO
H
H
H
OH
HO
H
HO
H
OH
H
CH2OPO32D-Psicose
1,6 bisphosphate
OH
CH2OPO32-
D-Tagatose
1,6 bisphosphate
H
OH
H
OH
CH2OPO32-
D-Sorbose
1,6 bisphosphate
Triosephosphate isomerase
DHAP
K eq 
GAP
DHAP
GAP
2
 4.7 x10
1

96
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.
TIM has an enediol intermediate
H
H
O
H
OH
H
H
C
OH
CH2OPO32-
H
C
OH
CH2OPO3
2-
C
OH
C
O
CH2OPO32-
GAP
enediol
DHAP
Transition state analogues Phosphoglycohydroxamate (A) and
2-phosphoglycolate (B) bind to TIM 155 and 100 times stronger
than GAP of DHAP
HO
B.
A.
OH
O
H
N
O
C
-
CH2OPO3
2-
O32-POH2C
O
O32-POH2C
O-
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
Geometry of the eneolate intermediate
prevents formation of methyl glyoxal
Orbital symmetry prevents double bond formation
needed for methyl glyoxal