1. Chapter 9
Enzyme Regulation

2. Metabolic Pathways
Regulation will depend on ability to alter flux thru the pathway by activation of the rate-limiting enzyme

3. Common Themes
Types of regulation depend upon the particular pathway and importance of pathway in the cell/tissue.
Negative feedback
Feed forward
Tissue specific isozymes

4. Mechanisms of Regulation
Regulation by compounds that bind reversibly to the active site
Regulation by alteration of the active site
Regulation by changing the concentration of the enzyme

5. Michaelis-Menton
Describes response of an enzyme to changes in substrate concentration
Powerful tool used to study normal and altered enzymes, such as those that produce diseases.
Like Hendersson-Hasselbach, LIVE IT< LOVE IT< LEARN IT.

6. Michaelis-Menten-2
The Reaction
The Dreaded Equation

7. Graph of Michaelis-Menton

8. Michaelis-Menten-3 Hyperbolic kinetics, saturation kinetics
[S]>>Km v=Vmax
[S]=Vmax v=Vmax/2
[S]<<Km velocity depends linearly on [S]

9. Lineweaver-Burk Plots

10. Warning Michaelis-Menten does not describe all enzymes
E.g. glucokinase
The model can not be used in situations in which [E]>[S]

11. Hexokinase Isozymes Catalyze the same reaction BUT
Different Km for glucose
Always remember that activity of enzymes will always depend upon the needs of a particular tissue.

12. Glucose Metabolism

13. Hexokinase I and Glucokinase
Hexokinase I RBCs
Km= 0.05 mM
Glucokinase liver
Km =5-6 mM

14. Glucokinase has a Higher Vmax
Prevents glucose from entering systemic circulation following carb-rich meal; minimizes hyperglycemia

15. MODY2 Maturity Onset Diabetes of the Young Type 2
Defect in pancreatic glucokinase
Associated with a reduction of level of insulin release for certain level of glucose

16. Velocity and Enzyme Concentration
Rate is directly proportional to the concentration of enzyme.

17. Inhibition within the Active Site
Inhibitors decrease rate of reactions
Inhibitors may be reversible or irreversible
Refer to Ch 8 on mechanism based inhibitors (irreversible)

18. Competitive Inhibition
Inhibitor COMPETES with substrate for the active site

19. Competitive Inhibition
Inhibitor can be diluted by increasing [S]
Vmax unchanged
Apparent Km increases
We needed to raise [S] to outcompete the inhibitor & saturate the enzyme

20. Graphical Depiction of Competitive Inhibition

21. Competitive Inhibition in Real Life
Al Martini and his alcohol dehydrogenase (ADH)
Ethanol + NAD+  Acetaldehyde NADH + H+
As more and more alcohol is being oxidized, the NADH/NAD+ ratio increases

22. NADH Inhibition of Enzymes
NADH competes with NAD+, thereby inhibiting ADH
Ethanol clearance from blood slows
NADH also inhibits enzymes involved in FA oxidation
Contributes to alcoholic fatty liver

23. Competitive Inhibition
Zocor and Lipitor inhibit HMG CoA reductase
Inhibits de novo cholesterol synthesis
Use of ethanol to treat ethylene glycol and methanol poisoning

24. Noncompetitive Inhibition
Binds to site other than active site or
Does not compete with a substrate for binding site

25. Noncompetitive Inhibition
Essentially no competition
Decreases available/effective enzyme concentration
Vmax decreases
Km unchanged if pure noncompetitive

26. Graphical Depiction of Noncompetitive Inhibition

27. Uncompetitive Inhibition
Reduce effective enzyme concentration
Vmax decreases
Inhibitor binds only ES
Km decreases

28. Regulation through Conformational Changes
Do not affect [E]
Respond quickly
Responsible for moment to moment regulation of activity
Mechanisms:
Allosteric
Reversible covalent modification
Control proteins

29. Allosteric Regulation
Regulation through binding of allosteric effectors
Bind to site separate from catalytic site (allosteric site)
Positive effectors activate enzyme
Negative effectors inhibit activity

30. Allosteric Inhibition
Allosteric modulators can indirectly alter the configuration of the active site, rendering the enzyme inactive
Noncompetitive inhibitors work by this mechanism

31. Allosteric Activation
An enzyme site may be activated sterically by an allosteric modulator

32. Properties of Allosteric Enzymes
Oligomeric = 2 or more subunits
Exist in 2 conformational states (R or T)
Exhibit cooperativity
Display sigmoid saturation curves

33. Activators and Inhibitors of Allosteric Enzymes
Activator binds R state
Inhibitor binds T state

34. Covalent Modification
MAJOR method for rapid and transient regulation of enzyme activity
Human genome encodes for > 1,000 different protein kinases
I guess protein phosphorylation is important!!

35. Phosphorylation & Dephosphorylation
ON and OFF Switch
Addition or removal of a phosphate group
Which amino acid residues get phosphorylated?
Serine, threonine or tyrosine

36. Protein kinases and phosphatases
Kinases add a phosphate
Phosphatases remove a phosphate

37. Muscle Glycogen Phosphorylase
Rate-limiting step in pathway of glycogen breakdown [glycogen  glucose 1-P]
Regulated by allosteric activator AMP AND
by phosphorylation

38. Protein-protein Interactions
Modulator proteins change shape of catalytic site or blocks the site
Calcium-calmodulin
Regulates a large # of proteins
G-proteins

39. Calcium-Calmodulin Neural impulse triggers calcium release from SR
Calcium binds calmodulin subunit of muscle glycogen phosphorylase kinase
Activated kinase then phosphorylates glycogen phosphorylase

40. Proteolytic Cleavage
Proenzymes = enzymes that must undergo proteolytic cleavage to be active
IRREVERSIBLE form of regulation
Zymogens = precursor proteins of proteases
Chymotrypsinogen, trypsinogen
Fibrinogen, prothrombin

41. Common Themes Concerning the Regulation of Metabolic Pathways
What are pathways?
Regulation occurs at Rate-Limiting Steps

42. Themes -2 Regulation matches function
Feedback Regulation- Negative Feedback
Feed-Forward Regulation
Counter- regulation
Keep opposing pathways separate
Compartmentation
Special needs
Limitation of substrate