1. Regulation of Metabolism
FCSN 543
Advanced Nutritional Biochemistry
Dr. David L. Gee

2. Characteristics of Regulatory Enzymes
Catalyze a rate-limiting step
Catalyze a committed step
Early step unique to a pathway
Irreversible step
Requires energy
Often results in a phosphorylated compound

3. Types of Regulatory Mechanisms
Non-covalent interactions
Covalent modifications
Changes in abundance of the enzyme

4. Non-covalent Interactions Substrate availability
Non-regulatory enzymes generally exhibit hyperbolic kinetics (Michaelis-Menton)
At low substrate concentration, reaction rate proportional to substrate concentration
Regulatory enzymes generally exhibit sigmoidal kinetics (positive cooperativity)
Changes of substrate concentrations at normal physiological levels greatly alter reaction rate

5. Non-covalent Interactions Allosteric Regulation
Binding of allosteric effectors at allosteric sites affect catalytic efficiency of the enzyme

6. Non-covalent Interactions Allosteric Regulation
Allosteric Activators
Decrease Km (increases the enzyme binding affinity)
Increases Vmax (increases the enzyme catalytic efficiency)

7. Non-covalent Interactions Allosteric Regulation
Allosteric Inhibitors
Increases Km (decreases enzyme binding affinity)
Decreases Vmax (decreases enzyme catalytic efficiency)

8. Molecues that act as allosteric effectors
End products of pathways
Feedback inhibition
Substrates of pathways
Feed-forward activators
Indicators of Energy Status
ATP/ADP/AMP
NAD/NADH
Citrate & acetyl CoA

9. Non-covalent Interactions Protein-Protein Interactions
Calmodulin (CALcium MODULted proteIN)
Binding of Ca++ to calmodulin changes its shape and allows binding and activation of certain enzymes

10. Binding of calcium to Calmodulin changes the shape of the protein
Unbound Calmodulin on left
Calcium bound Calmodulin on right. Stars indicate exposed non-polar ‘grooves’ that non-covalently binds proteins

11. Calmodulin Extracellular [Ca] = 5 mM Intracellular [Ca] = 10-4 mM
Most of Ca bound inside cells
Bound Ca can be released by hormonal action, nerve innervation, light, ….
Released Ca binds to Calmodulin which activates a large number of proteins

12. Calmodulin plays a role in:
Muscle contraction
Inflammation
Apoptosis
Memory
Immune response….
Metabolism
Activates phosphorylase kinase
Stimulates glycogen degradation during exercise

13. Types of Regulatory Mechanisms
Non-covalent interactions
Covalent modifications
Changes in abundance of the enzyme

14. Covalent Regulation of Enzyme Activity Phosphorylation and Dephosphorylation
Addition or deletion of phosphate groups to particular serine, threonine, or tyrosine residues alter the enzymes activity

15. Covalent Regulation of Enzyme Activity Limited Proteolysis
Specific proteolysis can activate certain enzymes and proteins (zymogens)
Digestive enzymes
Blood clotting proteins
Peptide hormones (insulin)

16. Covalent Regulation of Enzyme Activity Enzyme Cascades
Enzymes activating enzymes allows for amplification of a small regulatory signal

17. Types of Regulatory Mechanisms
Non-covalent interactions
Covalent modifications
Changes in abundance of the enzyme

18. Changes in Enzyme Abundance
Inducible vs Constitutive Enzymes
Induction is caused by increases in rate of gene transcription.
Hormones activate transcriptional factors
Increase synthesis of specific mRNA
Increase synthesis of specific enzymes

19. Hormones, Receptors, and Communication Between Cells
Intracellular receptors
lipid soluble hormones
Steroid hormones, vitamin D, retinoids, thyroxine
Bind to intracellular protein receptors
This binds to regulatory elements by a gene
Alters the rate of gene transcription
Induces or represses gene transcription

20. Hormones, Receptors, and Communication Between Cells Intracellular Receptors

21. Hormones, Receptors, and Communication Between Cells
Cell-surface receptors
Water soluble hormones
Peptide hormones (insulin), catecholamines, neurotransmitters
Three class of cell-surface receptors
Ligand-Gated Receptors
Catalytic Receptors
G Protein-linked Receptors

22. Hormones, Receptors, and Communication Between Cells
Ligand-gated receptors
Binding of a ligand (often a neurotransmitter) affects flow of ions in/out of cell
Gamma-amino butyric acid (GABA) binds and opens chloride channels in the brain
Valium (anti-anxiety drug) reduces the amount of GABA required to open the chloride channels

23. Hormones, Receptors, and Communication Between Cells Cell-Surface Receptors
Catalytic receptors
Binding of hormone activates tyrosine kinase on receptor which phosphorylates certain cellular proteins
Insulin receptor is a catalytic receptor with TYR Kinase activity

24. G-protein-linked receptors
Hormones, Receptors, and Communication Between Cells Cell-Surface Receptors
G-protein-linked receptors
Binding of hormone activates an enzyme via a G-protein communication link.
The enzymes produces intracellular messengers
cAMP
diacylglycerol (DAG))

25. Intracellular Messengers: Signal Transduction Pathways
Cyclic AMP (cAMP)
Diacylglycerol (DAG) & Inositol Triphosphate (IP3)
Cyclic GMP (cGMP)

26. G-Protein-Linked Receptors: The cAMP Signal Transduction Pathway
Two types of G-Proteins
Stimulating G protein (Gs)
Activate adenylate cyclase
Inhibitory G proteins (Gi)
Inhibit adenylate cyclase

27. G Proteins
G proteins are trimers
Three protein units
Alpha
Beta
gamma

28. Alpha proteins are different in Gs and Gi
Both have GTPase activity
Alpha proteins modify adenylate cyclase activity
AC stimulated by Alpha(s) when activated by a hormone
AC Inhibited by Alpha(I) when activated by other hormones

29. Family of G Proteins Binding of hormones to receptors causes:
GTP to displace GDP
Dissociation of alpha protein from beta and gamma subunits
activation of the alpha protein
Inhibition or activation of adenylate cyclase
GTPase gradually degrades GTP and inactivates the alpha protein effect (clock)

30. The cAMP Signal Transduction Pathway
cAMP – intracellular messenger
Elevated cAMP can either activate or inhibit regulatory enzymes
cAMP activates glycogen degradation
cAMP inhibits glycogen synthesis
[cAMP] affected by rates of synthesis and degradation
Synthesis by adenylate cyclase
Degradation by phosphodiesterase
Stimulated by insulin
Inhibited by caffeine

31. What does cAMP do? Activation of Protein Kinase A by cAMP
Activates or inhibits several enzymes of CHO and Lipid metabolism
Inactive form: regulatory+catalytic subunits associated
Active form: binding of cAMP disassociates subunits

32. DAG & IP3 Phosphotidylinositol Signal Transduction Pathway
Protein kinase C activated by DAG and calcium
Synthesis of DAG and IP3

33. cGMP The cGMP Signal Transduction Pathway
cGMP effects:
lowering of blood pressure & decreasing CHD risk
Relaxation of cardiac muscle
Vasodilation of vascular smooth muscle
Increased excretion of sodium and water by kidney
Decreased aggregation by platelet cells

34. cGMP The cGMP Signal Transduction Pathway
Two forms of guanylate cyclase
Membrane-bound
Activated by ANF (atrial natriuretic factor)
ANF released when BP elevated
Cytosolic
Activated by nitric oxide
NO produced from arginine by NO synthase
Nitroglycerine slowly produces NO, relaxes cardiac and vascular smooth muscle, reduces angina
cAMP activates Protein Kinase G
Phosphorylates smooth muscle proteins

35. cGMP The cGMP Signal Transduction Pathway