Regulation of Glycolysis

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Regulation of Glycolysis
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Learning Objectives
Describe substrate-limited and enzyme-limited
reactions.
Define or describe homeostasis, isozymes,
rate-limited reaction in a metabolic pathway.
Describe the allosteric regulation of
phosphofructokinase-1.
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A primary metabolic goal ATP constant
It is crucial for a cell to maintain the
concentration of ATP at a nearly constant
levelregardless of which fuel is used to produce
the ATP or the rate at which ATP is utilized.
Human metabolism is generally in a steady-state
a constant flux of nutrients which generates
energy and a constant release of waste
products. Changes in external circumstances
and/or the nutrient-energy supply can disturbe
this steady-state. These changes temporarily
affect the flux through metabolic pathways i.e.
steady-state concentrations of metabolites are
temporarily altered. This, in turn, triggers
regulatory mechanisms designed to return the
organism to steady-state to achieve homeostasis.
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homeostasis the maintenance of a dynamic steady
state by regulatory mechanisms that compensate
for changes in external circumstances.
Homeostasis of metabolic processes is time
dependent.
Metabolic requirements of an organism change with
time. Development and growth require more
nutrients than maintenance of the adult.
Development (and aging?) are timed processes
which vary with the individual, yet have a
certain basic pattern within each species.
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Regulatory Mechanisms
The flux through a metabolic pathway depends upon
the activities of the enzymes that catalyze each
reaction.
For biochemical reactions that are at or close to
equilibrium in the cell, the flux through this
reaction is substrate-limited the activity of
the enzyme is relatively high, and the substrate
equilibrates with product as fast as substrate is
supplied.
Other reactions are far from equilibrium. The
activity of the enzymes catalyzing these
reactions is low. The flux through these
biochemical reactions are enzyme-limited
substrate-availability is not rate-limiting.
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Enzyme-limited reaction
The rate is determined by the activity of the
enzyme. The flux through this reaction depends on
the extent of opening of an enzymatic valve.
Substrate-limited reaction
The flux is determined by the instantaneous
concentration of the substrate
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Enzymes speed up reaction rates. Thus, they
speed up the attainment of equilibrium. But if
the activity of an enzyme is low, the reaction
never gets even close to equilibrium the
substrate concentration may stay at a steady
state level, but this level is high product
formed is rapidly removed by the next step in the
pathway, and equilibration between substrate and
product does not occur.
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Every metabolic pathway has at least one reaction
that, in the cell, is far from equilibrium
because of the low activity of the enzyme. This
step is typically the rate-limiting step for the
entire metabolic pathway the flux through the
entire pathway is controlled by how much of this
enzyme is present, or how active the enzyme
is. In general, these rate-limiting step are very
exergonic, and essentially irreversible under
cellular conditions. They act as metabolic
valves, controlling the flux or flow of
intermediates through the entire pathway.
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Phosphofructokinase-1
fructose-6-phosphate ATP
fructose-1,6-bisphosphate ADP
The Keq for the phosphofructokinase-1 reaction
is about 250.
Under steady-state conditions in a typical cell
fructose-1,6-bisphosphate ADP
0.04
fructose-6-phosphate ATP
Therefore, this reaction is far from equilibrium.
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Phosphofructokinase-1 is under complex allosteric
regulation
Glucose-6-phosphate can go into several different
pathways
glycolysis pentose
phosphate pathway glycogen synthesis
blood glucose (liver)
The irreversible reaction catalyzed by
phosphofructokinase-1 is the step that commits a
cell to channeling glucose into glycolysis.
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E. coli phosphofructokinase-1
( 2 of the 4 identical subunits)
allosteric site (between subunits with ADP
bound)
active site
fructose-1,6-bisphosphate
ADP
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Allosteric inhibition of muscle PFK-1 by ATP
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Allosteric Regulation of phosphofructokinase-1
ATP allosterically inhibits PFK-1 ADP and AMP
bind at the allosteric site and relieve the
inhibition by ATP high citrate concentrations
increases the inhibitory effect of ATP
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Fructose-1,6-bisphosphatase-1 catalyzes the
conversion of fructose-1,6-bisphosphate to
fructose-6-phosphate in gluconeogenesis (the
reverse of the PFK-1 catalyzed reaction).
Fructose-2,6-bisphosphate activates PFK-1 and
inhibits FBP-1
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Fructose-2,6-bisphosphate is not an intermediate
in any metabolic pathway it is a regulatory
molecule.
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isozymes different proteins that catalyze the
same reaction.
LDH muscle (M) and heart (H) forms and mixtures
of the two in other cells hexokinase hexokinase
D (glucokinase) of liver and hexokinase of other
tissues, i.e. muscle glycogen phosphorylase
different forms in liver and muscle
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Hexokinase
Muscle hexokinase is allosterically inhibited by
its product, glucose-6-phosphate.
This adjusts the rate of glucose-6-phosphate
formation to bring it into balance with the rate
of its utilization, maintaining a steady-state
concentration.
Hexokinase has a low Km high affinity for
glucose.
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Liver hexokinase (hexokinase D, glucokinase)
Same reaction, but different regulatory
properties.
Liver glucokinase is allosterically inhibited by
fructose-6-phosphate.
Glucokinase has a high Km low affinity for
glucose.
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Pyruvate kinase
ATP allosterically inhibits pyruvate kinase by
decreasing its affinity for substrate
(phosphoenolpyruvate) Km increases. Acetyl-CoA
inhibits pyruvate kinase. Other fuels (fats and
amino acids) feed into acetyl-CoA and the TCA
cycle which can produce ample amounts of ATP.
When these fuels are available, acetyl-CoA slows
down the glycolytic flux until ATP levels drop.