Insights into Regulation

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Insights into Regulation

• Which way does the carbon go?

Ultimately, biochemistry seeks to learn how a
cell behaves how pathways of synthesis and
degradation are controlled and what determines
carbon flux. Obviously, a need for ATP must be
met by the cell making ATP, and ATP excess
likewise must deter ATP synthesis. A pathway in
a living system operates in a delicate balance
that favors one direction but is poised to
reverse or shutdown when a circumstances favor a
change. A search for how this is achieved leads
us directly into the realm of allosteric enzymes
and hormonal effects on cells. How these agents
change pathway direction is a fascinating insight
into the autonomous control of living systems.
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How are pathways regulated? Fortunately, we can
perhaps list 3 principal ways. First, because
many enzymes function at subsaturation with
regard to substrate, increasing the substrate
concentration by whatever means will increase the
velocity in a forward direction (click 1).
Second, many enzymes that regulate pathway flow
are allosteric. This brings T-state and R-state
transitions into the discussion and allows one to
focus on metabolites whose increase in
concentration could alter enzyme activity through
structural transitions (click 1). Third, is
covalent modification, which for our purposes,
mostly involves adding phosphate groups to an
enzyme, an action that can make the enzyme more
active or less active (click 1). Covalent
modification by phosphorylation is generally
under the control of hormones that activate c-AMP
which in turn activates protein kinase enzymes
(click 1). Click 1 to go on.
dB/dt
? velocity
A ? B
A
Allosteric effector
Protein kinase
cAMP
Hormone
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Phosphorylase
In the case of glycogen, allostery and covalent
modification play dominant roles in the
reglulation of enzyme activity. Allosteric
effects are seen in the T and R forms of glycogen
phosphorylase (click 1). This is not to be
confused with the a and b forms, which are
covalently modified by phosphate groups on the
protein (click 1) .
5-AMP
5-AMP
5-AMP
Active
Active
Active
Less active
Note, there are 4 different structural forms of
the dimeric protein phosphorylase. Click 1 to see
which forms are active. Both T-a and R-a are
active as well as R-b. R-b, however, depends on
5-AMP bound to the molecule, which depends on
the concentration of 5-AMP (click 1). T-a and
R-a stay active as long as phosphate is bound,.
R-b loses activity when 5-AMP dissociates and
T-a and R-a lose activity when phosphate is
cleaved.
A second point that should not escape your notice
is the position of the phosphate group in the R-a
(click 1). Phosphate is buried within the
protein and therefore more difficult to remove by
a phosphatase enzyme. In the T form the
phosphate group is exposed to the exterior of the
protein (click1). Click 1 to go on.
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Phosphatase
Phosphatases counter the action of kinases
by removing phosphate groups. These enzymes are
subject to covalent modification, which means
they too are controlled by hormones. Phosphatase
regulation, however, depends on the tissue. In
muscle, the phosphatase is bound to G protein,
which is the target of kinase enzymes (click 1).
The G protein has two sites for binding
phosphate. Filling site 1 activates the
phosphatase (click 1). Site 1 is filled by a
kinase that responds to insulin (click 1).
Alternatively, a c-AMP dependent protein kinase
fills both sites, either by acting directly on
the inactive complex (click 1) or just the second
site (click 1).
phosphatase
Active
Inactive
Both lead to an inactive phosphatase that
dissociates from the complex (click 1)
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Glycolysis-Gluconeogenesis
Glucose 6-PO4
Net carbon flux in glycolysis is controlled
mainly by PFK-1 (click 1). PFK-1 is sensitive to
allosteric effectors, the main one being fructose
2,6-bisPO4 (click 1). The reverse reaction is
catalyzed by fructose 1,6 bisphosphatase-1
(FBP-1) (click 1). High levels of the F2,6BP turn
on glycolysis by activating PFK-1 and inhibiting
FBP-1 (click 1). The result is a net flow of
carbon from G-6-P to pyruvate (click 1). If,
however, F2,6BP is destroyed by its phosphatase
(FBP-2), the flow towards pyruvate is halted in
intensity (click 1). A forced increase in the
carbon flux from pyruvate to G-6-P occurs during
gluconeogenesis (click 1). Stimulation occurs by
activating PEPCK by a c-AMP kinase (click 1).
This shows the importance of hormonal control,
specifically glucagon, in promoting
gluconeogenesis (click 1). Click 1 to go on.
Glucagon
F2,6BP
F2,6BP
c-AMP
PFK-1
FBP-1
PEPCK
PEP
OAA
Pyruvate
Malate
Fructose 2,6- bisPhosphate
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What have your learned?
1. Name a hormone that inhibits glycogen
breakdown. Describe the mechanism.
Insulin. Insulin stimulates phosphatases, one of
which converts phosphorylase a to phosphorylase b
2. Name an allosteric effector that stimulates
gluconeogenesis.
None. Stimulation by allostery occurs only in
glycolysis.
3. Predict the consequences to the cell by
activating PFK-2.
Glycolysis would be stimulated and
gluconeogenesis would be suppressed. PFK-2 is
the kinase that converts F6P into F2,6BP.

• What would be the expected effect of insulin on
  gluconeogenesis.
• Justify you answer.

Gluconeogenesis would be suppressed. One reasons
is that insulin activates phosphatases. PEPCK
would be a target for inactivation.