1. Enzyme Phosphorylation
Aulanni’am
Biochemistry Laboratory
Chemistry Department
Brawijaya University
Aulani " Biokimia Enzim" Presentasi 11

2. 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

3. Multiple ways of controlling enzyme activity
Control of enzyme availability
Amount of enzyme in the cell
Control of enzyme synthesis
Control of enzyme degradation
I could add examples as I go along for these situations. E.g, start out now saying there is tissue-specific expression of the gene for phosphorylase, etc. (instead of waiting until the last slide).
Location of enzyme in the cell
On/off membrane
Inside/outside organelle
Aulani " Biokimia Enzim" Presentasi 11

4. 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
Aulani " Biokimia Enzim" Presentasi 11

5. Aulani " Biokimia Enzim" Presentasi 11
“Feedback” control of metabolic pathways is often accomplished by allosteric mechanisms 1 2 3 4 5 6 A B C D E F G
(Excess G)
Aulani " Biokimia Enzim" Presentasi 11

6. Aulani " Biokimia Enzim" Presentasi 11
Allosteric modulation of aspartate transcarbamylase, the first unique step in the biosynthesis of pyrimidines (C, T, U)
Carbamoyl phosphate +
aspartate
ATCase
N-carbamoyl
aspartate
CTP
ATP
Aulani " Biokimia Enzim" Presentasi 11

7. The reaction catalyzed by aspartate transcarbamoylase
carbamoyl phosphate
H2PO4-
N-carbamoyl aspartate
Aulani " Biokimia Enzim" Presentasi 11

8. Aulani " Biokimia Enzim" Presentasi 11
Activity of aspartate transcarbamoylase (and many allosteric enzymes is cooperative with respect to aspartate
[aspartate] v0
Sigmoid (S-shaped) curve
Aulani " Biokimia Enzim" Presentasi 11

9. 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

10. Cooperativity of enzymatic activity: multiple states of the enzyme
ELA
EMA
(T state)
Less active
Lower affinity for S
(R state)
More active
Higher affinity for S
Aulani " Biokimia Enzim" Presentasi 11

11. What good is cooperativity in enzymatic activity?
(Same Vmax and v0 at 1/2 Vmax)
Steeper slope ensures greater sensitivity to variations in substrate concentration
Aulani " Biokimia Enzim" Presentasi 11

12. Effects of positive and negative modulators on cooperative enzymes
ATP and CTP are positive and negative modulators of aspartate transcarbamoylase
[substate] v0
positive
negative
Aulani " Biokimia Enzim" Presentasi 11

13. Aulani " Biokimia Enzim" Presentasi 11
Allosteric activators and inhibitors stabilize different states of the enzyme (ATCase)
ELA
EMA
(T state)
Less active
Lower affinity for S
Stabilized by CTP
(R state)
More active
Higher affinity for S
Stabilized by ATP
Aulani " Biokimia Enzim" Presentasi 11

14. Aulani " Biokimia Enzim" Presentasi 11
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)
Aulani " Biokimia Enzim" Presentasi 11

15. 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

16. 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
I said it’ not clear if you call this allosteric or not.
Aulani " Biokimia Enzim" Presentasi 11

17. 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

18. > Modification of thrombin activity by thrombomodulin clot Thrombin
Protein C
Thrombomodulin
Protein C zymogen
(contains Gla)
Aulani " Biokimia Enzim" Presentasi 11

19. 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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20. Modulation by phosphorylation
kinase
ATP
ADP
phosphatase
H2O Pi
PO32-
Enzyme X (active)
Enzyme X (inactive)
Phosphorylation may also result in inactivation,
depending on the enzyme
Aulani " Biokimia Enzim" Presentasi 11

21. 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
Any other cool examples of irreversible modification? Maybe a ubiquitin-dependent degradation?
Aulani " Biokimia Enzim" Presentasi 11

22. 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

23. Aulani " Biokimia Enzim" Presentasi 11
to be continued .
Aulani " Biokimia Enzim" Presentasi 11