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Enzyme Phosphorylation
Aulanni’am
Biochemistry Laboratory
Chemistry Department
Brawijaya University
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Enzyme regulation: How to control the
activity of a pathway in response to
the needs of the cell or body
Aulani " Biokimia Enzim" Presentasi
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Multiple ways of controlling
enzyme activity
• Control of enzyme availability
–Amount of enzyme in the cell
•Control of enzyme synthesis
•Control of enzyme degradation
–Location of enzyme in the cell
•On/off membrane
•Inside/outside organelle
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Multiple ways of controlling
enzyme activity
• Control of enzyme activity
– Allosteric activation or inactivation: interaction of
small molecule with enzyme, but not at active site
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“Feedback” control of metabolic pathways
is often accomplished by allosteric
mechanisms
1
A
2
B
3
C
4
D
5
E
(Excess G)
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6
F
G
Allosteric modulation of aspartate transcarbamylase, the
first unique step in the biosynthesis of pyrimidines (C,
T, U)
Carbamoyl
phosphate ATCase N-carbamoyl
+
aspartate
aspartate
ATP
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CTP
The reaction catalyzed by aspartate
transcarbamoylase
NH 2
C
O
OPO 3=
aspartate
carbamoyl phosphate
H2PO4-
N-carbamoyl aspartate
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Activity of aspartate transcarbamoylase (and
many allosteric enzymes is cooperative with
respect to aspartate
v0
Sigmoid (Sshaped) curve
[aspartate]
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Cooperativity in enzymatic activity
• Multimeric enzymes
• Binding of S to one protomer causes change in
adjacent protomer, increasing the affinity of both
protomers for the substrate
Aulani " Biokimia Enzim" Presentasi
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Cooperativity of enzymatic activity:
multiple states of the enzyme
ELA
(T state)
Less active
Lower affinity for S
EMA
(R state)
More active
Higher affinity for S
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What good is cooperativity in enzymatic
activity?
(Same Vmax and
v0 at 1/2 Vmax)
v0
[S]
Steeper slope ensures greater sensitivity to
variations in substrate concentration
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Effects of positive and negative modulators
on cooperative enzymes
ATP and CTP are positive and negative modulators of
aspartate transcarbamoylase
positive
v0
negative
[substate]
Aulani " Biokimia Enzim" Presentasi
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Allosteric activators and inhibitors
stabilize different states of the enzyme
(ATCase)
ELA
(T state)
Less active
Lower affinity for S
Stabilized by CTP
EMA
(R state)
More active
Higher affinity for S
Stabilized by ATP
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Allostery is accomplished by conformational
changes in ATCase and other enzymes
Red: upper catalytic
trimer
Blue: lower catalytic
trimer
Green: regulatory
dimers (bind ATP and CTP)
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Other about allosterism
• Allosteric modulators can change Km or Vmax or
both
• Not all allosteric enzymes are cooperative
• Allosteric modulation renders an enzyme
sensitive to metabolites other than its own
substrates and products (e.g., ATCase is
sensitive to endproducts of purine and
pyrimidine biosynthesis).
Aulani " Biokimia Enzim" Presentasi
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Multiple ways of controlling
enzyme activity
• Control of enzyme activity
– Allosteric activation or inactivation:
interaction of small molecule with enzyme,
but not at active site
– Interaction with a large molecule, usually
another protein
Example: Thrombin and thrombomodulin
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Modification of thrombin activity by
thrombomodulin
• Thrombomodulin (on cell surface of vascular
endothelium)
– Decreases activity of thrombin toward
fibrinogen
– Increases ability of thrombin to activate
Protein C, an inhibitor of clotting
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Modification of thrombin activity by
thrombomodulin
>
Thrombin
Protein C
Thrombomodulin
Protein C zymogen
(contains Gla)
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clot
Multiple ways of controlling
enzyme activity
• Control of enzyme activity
– Allosteric activation or inactivation
– Interaction with another protein
– Reversible covalent modification
Example: phosphorylation
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Modulation by phosphorylation
ATP
kinase
ADP
PO32-
Enzyme X
(inactive)
phosphatase
H 2O
Pi
Enzyme X
(active)
Phosphorylation may also result in inactivation,
depending on the enzyme
Aulani " Biokimia Enzim" Presentasi
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Multiple ways of controlling
enzyme activity
• Control of enzyme activity
– Allosteric activation or inactivation
– Interaction with another protein
– Reversible covalent modification
– Irreversible modification
Example: zymogen activation
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Multiple ways of controlling a single enzyme:
Glycogen phosphorylase
• Gene expression
– Multiple genes, tissue-specific expression
– Rates transcription vary
• Protein activity
–Allosteric control
–Reversible phosphorylation
Aulani " Biokimia Enzim" Presentasi
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Aulani " Biokimia Enzim" Presentasi
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