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Aulani " Biokimia Enzim" Presentasi 11
Enzyme Phosphorylation
Aulanni’amBiochemistry LaboratoryChemistry DepartmentBrawijaya University
Aulani " Biokimia Enzim" Presentasi 11
Enzyme regulation: How to control the activity of a pathway in response to
the needs of the cell or body
Aulani " Biokimia Enzim" Presentasi 11
Multiple ways of controllingenzyme 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
Aulani " Biokimia Enzim" Presentasi 11
Multiple ways of controllingenzyme activity
• Control of enzyme activity– Allosteric activation or inactivation: interaction of
small molecule with enzyme, but not at active site
Aulani " Biokimia Enzim" Presentasi 11
“Feedback” control of metabolic pathways is often accomplished by allosteric
mechanisms
A B C D E F G1 2 3 4 5 6
(Excess G)
Aulani " Biokimia Enzim" Presentasi 11
Carbamoylphosphate
+aspartate
N-carbamoylaspartate
ATCaseCTP
Allosteric modulation of aspartate transcarbamylase, the first unique step in the biosynthesis of
pyrimidines (C, T, U)
ATP
Aulani " Biokimia Enzim" Presentasi 11
The reaction catalyzed by aspartate transcarbamoylase
carbamoyl phosphateaspartate
N-carbamoyl aspartate
H2PO4-
C
NH2
O OPO 3=
CO-O
CH2
CHCOO -N
H
C
NH2
O
Aulani " Biokimia Enzim" Presentasi 11
Activity of aspartate transcarbamoylase (and many allosteric enzymes is cooperative with
respect to aspartate
[aspartate]
v0Sigmoid (S-shaped) curve
Aulani " Biokimia Enzim" Presentasi 11
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 11
Cooperativity of enzymatic activity: multiple states of the enzyme
ELA EMA
(T state)Less activeLower affinity for S
(R state)More activeHigher affinity for S
Aulani " Biokimia Enzim" Presentasi 11
What good is cooperativity in enzymatic activity?
v0
[S]
(Same Vmax and v0 at 1/2 Vmax)
Steeper slope ensures greater sensitivity to variations in substrate concentration
Aulani " Biokimia Enzim" Presentasi 11
ATP and CTP are positive and negative modulators of aspartate transcarbamoylase
[substate]
v0
positive
negative
Effects of positive and negative modulators on cooperative enzymes
Aulani " Biokimia Enzim" Presentasi 11
Allosteric activators and inhibitors stabilize different states of the enzyme
(ATCase)
ELA EMA
(T state)Less activeLower affinity for SStabilized by CTP
(R state)More activeHigher affinity for SStabilized by ATP
Aulani " Biokimia Enzim" Presentasi 11
Allostery is accomplished by conformational changes in ATCase and other enzymes
Red: upper catalytictrimer
Blue: lower catalytictrimer
Green: regulatorydimers (bind ATP and CTP)
Aulani " Biokimia Enzim" Presentasi 11
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 11
Multiple ways of controllingenzyme 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
Aulani " Biokimia Enzim" Presentasi 11
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
Aulani " Biokimia Enzim" Presentasi 11
Modification of thrombin activity by thrombomodulin
clot
Thrombin
Protein C
>
Thrombomodulin
Protein C zymogen(contains Gla)
Aulani " Biokimia Enzim" Presentasi 11
Multiple ways of controllingenzyme activity
• Control of enzyme activity
– Allosteric activation or inactivation
– Interaction with another protein
– Reversible covalent modification
Example: phosphorylation
Aulani " Biokimia Enzim" Presentasi 11
Modulation by phosphorylation
Enzyme X(inactive)
PO32-
Enzyme X(active)
Phosphorylation may also result in inactivation,depending on the enzyme
kinaseATP ADP
phosphataseH2OPi
Aulani " Biokimia Enzim" Presentasi 11
Multiple ways of controllingenzyme activity
• Control of enzyme activity
– Allosteric activation or inactivation
– Interaction with another protein
– Reversible covalent modification
– Irreversible modification
Example: zymogen activation
Aulani " Biokimia Enzim" Presentasi 11
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 11