Transcript glycolysis

Glycolysis
Andy Howard
Introductory Biochemistry
25 March 2008
25 Mar 2008
What we’ll discuss

Glycolysis
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Overview
Steps through
TIM
Steps to
pyruvate
Fate of pyruvate
Glycolysis
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Glycolysis
(continued)
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Free energy
Regulation
Other sugars
Entner-Doudoroff
Pathway*
p. 2 of 56
25 Mar 2008
Glycolysis
Now we’re ready for the specifics of
metabolism
 Why glycolysis first?

Well-understood (?) early on
 Illustrates concepts used later
 Inherently important

Glycolysis
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The big picture
Conversion of glucose to pyruvate
 Catabolic, ten steps, energy-yielding
 Overall reaction:

glucose + 2 ADP + 2 NAD+ + 2Pi 
2pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
Glycolysis
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Significance
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Why is this important?
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Energy production
(ATP and NADH)
Pyruvate as precursor to various
metabolites
Some steps require energy
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So it isn’t all energy-yielding
The net reaction yields energy
Glycolysis
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The reactions
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See fig. 11.2 and the table in the
HTML notes
Wide variety of enzyme sizes
Most structures have been
determined by X-ray crystallography
Glycolysis
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The pathway through TIM
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Fig. courtesy U.Texas
Glycolysis
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Pathway to pyruvate
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Bottom half of same graphic
Glycolysis
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G-6-P
Hexokinase
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Transfers γ-phosphoryl group of
ATP to oxygen atom at C-6 of
glucose, producing glucose 6phosphate and ADP.
Coupling between ATP hydrolysis
and an energy-requiring reaction is
very close: phosphate is transferred
directly from ATP to the recipient
molecule, in this case glucose.
The reaction catalyzed by
hexokinase is energetically favored:
Go’ = -22.3 kJ/mol
Glycolysis
p. 9 of 56
PDB 2YHX
Yeast
52kDa
monomer
25 Mar 2008
Hexokinase isozymes
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various isozymes (functionally related
but structurally slightly distinct) forms
of hexokinase in humans
liver form has Km in millimolar range,
perhaps a factor of 1000 higher than
the Km of hexokinase found in other
tissue
Liver form is therefore much less
active than the other forms unless the
liver glucose concentration is high
Glycolysis
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25 Mar 2008
Activity and complexity
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Hexokinase is active on sugars besides
glucose;
activity against mannose is comparable
to the activity on glucose
Hexokinase has the highest molecular
mass per monomer of any of the
glycolytic enzymes; given that it is the
first enzyme in an important pathway, it
makes sense that it is large and complex.
Glycolysis
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25 Mar 2008
Phosphoglucomutase
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Interconverts phosphorylated
forms of glucose—
glucose 1-P and glucose 6-P.
Intermediate is
bisphosphorylated
equilibrium between the 1-P and
6-P forms is determined by
relative concentrations.
Active on other phosphorylated
aldoses in addition to glucose.
This enzyme doesn’t appear on
the chart:
not part of the linear pathway
from glucose to pyruvate.
Glycolysis
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PDB 1ZOL
Lactococcus
24 kDa monomer
25 Mar 2008
Glucose 6-phosphate
isomerase
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F-6-P
interconverts two monophosphorylated sugars—
glucose 6-phosphate and fructose 6-phosphate.
Interconversion proceeds through (1,2) ene-diol
intermediate
with enzyme present the energy barriers around
this ene-diol are lowered enough to speed the
interconversion.
Also called phosphohexoseisomerase or
phosphoglucose isomerase
Glycolysis
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Properties of G6P isomerase
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Dimeric enzyme plays roles
extracellularly as well as
intracellularly: it can
function as a nerve growth
factor.
Each monomer contains
two unequal-sized
domains, and the active
site is formed by the
association of the two
subunits.
Glycolysis
PDB 1U0F
mouse
124 kDa dimer
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Phosphofructokinase-1
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catalyzes phosphorylation at the 1
position of fructose 6-phosphate.
F-1,6-bisP
example of a kinase that acts on an
already-phosphorylated form, creating
a bisphosphorylated compound.
ADP sometimes acts as an allosteric
activator on this enzyme as well as
being a product of the reaction.
We’ll discuss PFK-2 later
Glycolysis
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PFK-1
structures
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Of all the enzymes in this
pathway it appears to be
the one for which the least
structural information is
available
Best structure determined
to date for the allosteric
enzyme was Phil Evans's
2.4 Å structure from 1988,
and there have not been
many other structures
done.
Glycolysis
PDB 4pfk
E.coli
140 kDa tetramer
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Lactobacillus
PFK
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This one isn’t allosteric
No MgADP binding
observed (> 20 mM)
Yet it’s highly
homologous!
Effector binding site is
very different
Glycolysis
PDB 1zxx
Lactobacillus bulgaricus
140 kDa tetramer
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+
Aldolase
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Catalyzes actual C-C bond cleavage:
fructose 1,6-bisphosphate 
D-glyceraldehyde-1-phosphate +
dihydroxyacetone phosphate
large and important enzyme
Some bacterial and yeast forms require a
divalent cation as a cofactor;
eukaryotic aldolases do not.
The non-cationic forms proceed through an
imine (Schiff-base) intermediate.
Glycolysis
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Secondary
activity
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Enzyme is active on
fructose 1-phosphate as
well as its "standard"
substrate, fructose 1,6bisphosphate; in this
context it forms part of
catabolic pathway by
which fructose itself can
be used as an energy and
carbon source.
Glycolysis
PDB 1zah
Rabbit muscle
80 kDa dimer
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Triosephosphate
isomerase
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Interconverts two 3-C
phosphosugars
possibly the most
efficient enzyme
known, in terms of the
rate acceleration
afforded by the
enzyme relative to the
uncatalyzed reaction.
Glycolysis
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TIM Barrels
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TIM is an enzyme with a
characteristic structure in
which alpha helical
stretches alternate with
beta strands such that the
beta strands curve around
to form a barrel-like
structure with the helices
outside.
This structural motif
appears in many other
enzymes, and has become
known as a "TIM barrel."
Glycolysis
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PDB 1YPI
Saccharomyces
27 kDa monomer
25 Mar 2008
Glyceraldehyde 3phosphate dehydrogenase
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medium-sized dimeric
or tetrameric enzyme
responsible for the
conversion of Glyc-3P
to 1,3bisphosphoglycerate.
Somewhat allosteric
Glycolysis
PDB 1GD1
Bacillus
stearothermophilus
74 kDa dimer
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Phosphoglycerate
kinase
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Glycolysis
catalyzes dephosphorylation of
1,3-bisphosphoglycerate to 3phosphoglycerate with production
of ATP from ADP
named for reaction running in
opposite direction relative the one
shown in chart.
In the direction shown in the table it
produces ATP rather than
consuming it.
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PGK Structural
Notes
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Has a hinge motion about a
point near the center of the
molecule; the open and
closed forms of the enzyme
involve movements as large
as 17Å in the residues
farthest from the hinge point.
Enzyme is primarily alphahelical in conformation.
Glycolysis
PDB 16pk
Trypanosoma brucei
184 kDa tetramer;
monomer shown
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Phosphoglycerate
mutase
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interconverts 3phosphoglycerate and 2phosphoglycerate
Mechanism of reaction
involves formation of 2,3bisphosphoglycerate via
transient phosphorylation
of a histidine residue of
the enzyme.
Glycolysis
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2-phosphoglycerate
PDB 1e59
E.coli
55 kDa dimer;
monomer shown
25 Mar 2008
PG Mutase:
a problem!
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2,3BPG can diffuse from
phosphoglycerate mutase,
2,3however, leaving the enzyme
bisphosphoglycerate
trapped in an unusable state.
Cells make excess 2,3BPG
(using the enzyme
bisphosphoglycerate mutase) in
order to drive 2,3BPG back to
phosphoglycerate mutase, so
the reaction can go to
completion.
Glycolysis
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Enolase
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interconverts 2phosphoglycerate &
phosphoenolpyruvate
This reaction plays a
role in
gluconeogenesis as
well as glycolysis.
PDB 4enl
Saccharomyces
97 kDa dimer; monomer shown
Glycolysis
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Enolase details
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Mg2+ ions are required for activity, at
least in some forms of the enzyme.
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Vertebrate genes code for two slightly
different forms of the monomer of
enolase, alpha and beta.
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Most of the enolase in fetal tissue is
alpha-alpha; mature skeletal muscle
contains beta-beta; some alpha-alpha
remains in smooth muscle tissue.
Glycolysis
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Pyruvate Kinase
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transfers a phosphate
from
phosphoenolpyruvate to
ADP, producing pyruvate
and ATP
The reaction is essentially
irreversible
(Go’ ~ -30 kJ mol-1)
Fructose 1,6bisphosphate, the
substrate for the aldolase
reaction, is a feed-forward
activator
of the reaction
Glycolysis
PDB 1PKM
Cat muscle
236 kDa tetramer
monomer shown
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So we’ve gotten
to pyruvate
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This is conventionally seen as the
endpoint of glycolysis
It’s worthwhile, though, to see what can
happen to the products
Pyruvate (memorize that structure!) is an
important intermediate in several
pathways
Glycolysis
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What happens to pyruvate?
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Four paths:
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Pyruvate + CoASH  acetylCoA + CO2;
this leads to Krebs cycle, to fatty acid
biosynthesis, and amino acids
Pyruvate + CO2  oxaloacetate;
this is an anapleurotic mechanism for Krebs
cycle
Pyruvate + NADH + H+  lactate + NAD+
Pyruvate + H+  acetaldehyde + CO2
acetaldehyde + NADH + H+  ethanol + NAD
Glycolysis
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Pyruvate to Lactate
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Lactate dehydrogenase catalyzes
pyruvate + NADH + H+  lactate + NAD+
Occurs in some anaerobic bacteria and in
mammals (e.g. in muscles) if oxygen is not
plentiful: anaerobic glycolysis
Net glycolysis reaction under these conditions:
glucose + 2 Pi2- + 2 ADP3- 
2 lactate- + 2 ATP4- + 2H2O
Can result in drop in blood pH until reverse
reaction (in liver) restores pH and regenerates
pyruvate
Glycolysis
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Lactate
dehydrogenase
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Typical tetrameric
Rossmann-fold NADdependent dehydrogenase
Structural homology to
other NAD-binding
enzymes
Glycolysis
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PDB 1xiv
Plasmodium
140 kDa tetramer
25 Mar 2008
Pyruvate to ethanol
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Pyruvate decarboxylated to acetaldehyde:
pyruvate + H+  acetaldehyde + CO2
Acetaldehyde is reduced to ethanol:
acetaldehyde + NADH + H+ 
ethanol + NAD
Net glycolytic reaction is
glucose + 2 Pi2- + 2 ADP3- + 2H+ 
2 ethanol + 2CO2 + 2 ATP4- + 2H2O
Yeast depend on this pathway
Glycolysis
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Pyruvate
decarboxylase
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Catalyzes first reaction in
pathway to ethanol
TPP-dependent reaction: see
section 7.7, especially fig. 7.15
Related to the pyruvate
dehydrogenase complex that
we will meet in chapter 13
Glycolysis
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PDB 1pvd
Saccharomyces
62 kDa monomer
25 Mar 2008
Alcohol
dehydrogenase
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Second reaction
in fermentation path
Reaction itself is reversible:
ethanol  acetaldehyde
direction leads to detox in
humans
Often unselective: can be
used to oxidize other primary
alcohols
Glycolysis
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PDB 2hcy
Saccharomyces
156 kDa tetramer
25 Mar 2008
Free energy in
glycolysis
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Cliché:
G matters, not Go’!
See fig. 11.11:
Several reactions are endergonic as
far as Go’ are concerned, but they’re
flat or exergonic with G.
Glycolysis
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Hamori’s data
Step
Reactant
0
1
2
3
4
5
6
7
8
9
Glucose, ATP
G6P
F6P+ATP
FDP
Glyc3P+NAD+Pi+ADP
3PG
PG2
PEP+ADP
PYR+NADH
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Products
DGo'
G6P, ADP
F6P
FDP + ADP
2 Glyc-3P
3PG+ATP+NADH
2PG
PEP
PYR+ATP
Lac+NAD
0
-5.1
0.49
-4.3
7.4
-6.5
2.1
-1.3
-12.2
-11.9
DG
Cum DGo'
0
-9.5
-0.06
-6.2
-0.17
-0.56
-0.27
-0.64
-7.4
0
0
-21.3
-19.3
-37.3
-6.3
-33.5
-24.7
-30.2
-81.2
-131.0
Sum DG
0
-39.7
-40.0
-65.9
-66.7
-69.0
-70.1
-72.8
-103.8
-103.8
E. Hamori (1975) J.Chem.Ed. 52: 370
Individual values in kcal mol-1
Cumulative values in kJ mol-1
Glycolysis
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My version of fig. 11.11
Standard and actual free energy
0
0
1
2
3
4
5
6
7
8
9
10
Cumulative free energy changes, kJ mol-1
-20
-40
Cum DGo'
Sum DG
-60
-80
-100

-120
Data from Hamori (1975),
J.Chem.Ed.52:370
-140
Step in glycolysis
Glycolysis
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Which steps are irreversible?
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Just three:
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Glucose to G-6P (G ~ -40 kJ mol-1)
Fructose-6-P to Fructose-1,6-bisP (-26)
PEP to pyruvate (-31)
All the others are reversible
So the controls are likely to be at those
three points: and they are!
Glycolysis
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Regulation of glycolysis
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Two ways to study this:
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Enzymology (know thy enzymes)
Metabolic biochemistry (know concentrations
and fluxes under cellular conditions)
Sometimes enzymology gives interesting
but cellularly unrealistic results (e.g.,
inhibitors that only inhibit at 100 * actual
cellular concentrations)
Glycolysis
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Regulators of glycolysis
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See fig. 11.12:
Glucose-6-P inhibits hexokinase
ATP and citrate inhibit PFK-1
AMP, Fructose 2,6-bisP activate PFK1
F 1,6-bisP activates pyruvate kinase
ATP inhibits pyruvate kinase
Glycolysis
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Control at the transport level
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[glucoseintracellular] < [glucoseblood]
(except in liver);
passive transport aided by transporters
All mammalian cells have transporters
Na+ dependent cotransport: SGLT1
in intestinal & kidney cells
GLUT family (1-7) found in other cells
Glycolysis
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Insulin and Glut4 (fig. 11.13)
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When insulin binds to tyr-kinase receptors, they
dimerize and promote fusion of intracellular
vesicles with the plasma membrane
Vesicles carry Glut4 transporters
This happens only in striated muscle and
adipose tissue—that’s where the Glut4
transporters are
This is only one of several roles that insulin plays
in glucose and lipid metabolism
Glycolysis
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How does glucose get in?
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SGLT1 and GLUT4 stories (above)
GLUT1,3 provide basal intake levels
GLUT2 brings glucose in & out of liver
GLUT5: fructose in small intestine
GLUT7: G6P from cytoplasm to ER
Doesn’t stay neutral long:
once it gets into the cell, it gets 6phosphorylated with help of hexokinase
Glycolysis
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Regulation of hexokinase
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Isozymes I,II,III: Km ~ 0.1mM;
G6P allosterically inhibits the enzyme
Glucokinase (IV): unregulated, high Km
… found in liver & islet cells
Pileup of G6P occurs if downstream
steps are inhibited;
allostery in hexokinase I-III alleviates that
Glycolysis
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GKRP and F-6P: regulators
of liver glucokinase
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Glucokinase regulatory protein binds
glucokinase in presence of F-6-P and F-1-P
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Lowers affinity to ~ 10mM
sigmoidal kinetics
With high [glucose], GKRP pulls GK into
nucleus; low [glucose] makes GKRP release
GK so it can phosphorylate glucose
Glycolysis
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Regulation of PFK-1
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Nucleotides:
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ATP is both substrate and
(usually) allosteric inhibitor
ATP increases apparent Km for F6P
AMP is activator: relieves ATP inhibition
ADP’s effects vary
[ATP] fairly constant; [AMP] varies
Citrate (Krebs cycle component) inhibits it
[H+] is also an inhibitor (lactic acid debt)
Glycolysis
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F-2,6-bisP and
PFK-1, PFK-2
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Potent activator of PFK-1
Absent in prokaryotes
F-2,6-bisP Formed by action of PFK-2
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Fructose 2,6-bisphosphate
(n.b.: drawn backward from text)
ATP + F-6-P  F-2,6-bisP + ADP
Stimulated by Pi, inhibited by citrate
Same enzyme is also fructose 2,6bisphosphatase at different active site
See fig. 11.16!
Glycolysis
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PFK-2 and glucagon
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High [glucagon] turns on adenylyl
cyclase pathway in liver
Protein kinase A then
phosphorylates a serine in PFK-2
That turns on phosphatase activity,
turns off PFK-2 activity
Thus [F-2,6-bisP] , PFK-1 less Phosphofructokinase-2
active, glycolysis is depressed
PDB 2AXN
57 kDa monomer
Glycolysis
p. 50 of 56
25 Mar 2008
What if glucose is being
rapidly metabolized?
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[glucagon] , [F-6-P], [F-2,6-bisP]
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F-6-P is a substrate for PFK-2
F-6-P is a potent inhibitor of F-2,6bisphosphatase
That activates a phosphatase that
dephosphorylates PFK-2
PFK-2 activity , phosphatase activity  !
See figure 11.17
Glycolysis
p. 51 of 56
25 Mar 2008
Pyruvate kinase regulation
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Four isozymes in mammals
Liver, kidney, blood forms have sigmoidal
kinetics for [PEP]
Activated by F-1,6-bisP, inhibited by ATP
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Low [F-1,6-bisP]:
ATP almost completely inhibits enzyme
High [F-1,6-bisP]: ATP almost irrelevant
Feed-forward activation
Glycolysis
p. 52 of 56
25 Mar 2008
Pyruvate kinase,
phosphorylation, and glucagon
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One isozyme (liver, intestine) is sensitive
to [glucagon]:
Protein kinase A (see PFK-2!)
phosphorylates pyruvate kinase,
inactivating it somewhat
Glucagon stimulates protein kinase A, so
it tends to inactivate pyruvate kinase
Glycolysis
p. 53 of 56
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Pasteur effect
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Definition: increase in glycolysis under
anaerobic conditions
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Relevant to yeast behavior
Also to muscle metabolism when exercising,
since not enough [O2] is getting to the muscles to
maintain oxidative phosphorylation
Reason: less ATP per glucose molecule with
anaerobic metabolism, so you need to use more
glucose to get the same amount of ATP out
Modulation at PFK-1 level, others
Glycolysis
p. 54 of 56
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Fructose

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Transported with GLUT5
Ordinarily phosphorylated to F-1-P by ATPdependent fructokinase
F-1-P cleaved to DHAP and glyceraldehyde
by fructose 1-P aldolase
Glyceraldehyde is 3-phosphorylated by ATPdependent triose kinase
DHAP, Glyc-3-P then enter glycolysis as
usual
Glycolysis
p. 55 of 56
25 Mar 2008
Fructosemetabolizing
enzymes

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Fructokinase
Fructokinase
F-1P aldolase
PDB 2hlz
(now considered a subset human
of ordinary F-1,6-bisP
136 kDa tetramer
aldolase)
Triose kinase (no structures
yet!)
Glycolysis
p. 56 of 56
25 Mar 2008