1. Preparing for the Exam
Question 1:  Amino acid structures and abbreviations.
Question 2:  Mechanism of trypsin.
Question 3:  Serine protease evolution and function.
Question 4:  Non-covalent inhibition of Protein Function
Question 5:  Purification and analysis of a protein
Question 6:  Michaelis-Menton kinetics
Question 7:  Reaction coordinates and enzyme catalyzed reactions
Question 8:  Regulation of Protein Function
Question 9:  Protein folding
Question 10:  IMFs
Question 11:  Lysozyme mechanism
Question 12:  Hemoglobin and the Bohr Effect
Question 13: RNAse A Mechanism

2. Drug Design Example: HIV Protease
Transition State Analogs

3. Example Acid-Base Catalysis: RNase A
Enzyme responsible for degrading RNA in the cell

4. Example Acid-Base Catalysis: RNase A

5. Example Acid-Base Catalysis: RNase A
1) The different protonation states of two active site histidines (His12 and His199) (See PDB Entry 4AO1)
His12 pKA=4.72
His119 pKA=6.03

6. Example Acid-Base Catalysis: RNase A
His12: Expected to be fully deprotonated
His119: Expected to be partially protonated
His12 pKA=4.72
His119 pKA=6.03

7. Example Acid-Base Catalysis: RNase A
So how does this difference in pKA arise?

8. Example Acid-Base Catalysis: RNase A
Substrate Binding: Orientation Effects
Look at the Coulombic surface
What is the charge of RNA?
Where would the phosphate backbone bind most productively?
Where does that binding mode place the C2’ hydroxyl group of the 5’ ribose ring?
Which direction does the RNA substrate run in the binding site?

9. Example Acid-Base Catalysis: RNase A
Look at the Coulombic surface
What is the charge of RNA?
Where would the phosphate backbone bind most productively?
Where does that binding mode place the C2’ hydroxyl group of the 5’ ribose ring?
Which direction does the RNA substrate run in the binding site?
By looking at an appropriate representation of the protein and knowing the enzyme-catalyzed reaction, you are able to answer critical mechanistic questions about an enzyme you only recently knew existed

10. Protein Regulation
There are three basic mechanisms to control/regulate protein function
Localization of the protein and/or its partners
Binding of effector molecules or Posttranslational Modifications
Physical amounts of protein and the lifetime of each molecule
This is the simplest mechanism to control protein function

11. 1. Regulation by Localization
Signal sequences target protein to specific compartment
Covalent modifications force protein to stay in a specific compartment
Interactions with other proteins that exist in a specific compartment

12. 2. Regulation by Effector Molecules
Usually induce conformational changes that alter protein function
Inhibitors, cooperativity
Allostery
Alternate binding sites that promote or weaken subsequent binding events when occupied
Covalent modifications
Phosphorylation, methylation, glycosylation, acylation, acetylation, etc.

13. 2. Regulation by Effector Molecules – Covalent Modification

14. 3. Protein Regulation by Degradation (Lifetime)
The lifetime of a protein is a simple method of regulation
Proteins involved in cell cycle or metabolism generally have short lifetimes. Why?
Structural proteins (actin/histones) have longer lifetimes.
How is a protein’s lifespan determined?
Intrinsic stability of the tertiary structure
Certain recognition sequences
Proteins are degraded in the Proteasome

15. 1. Regulation by Localization
In eukaryotic cells, proteins can be targeted to specific locales: ER, Golgi, Nucleus, mitochondrion or secreted
Specific signal sequences interact with other proteins at the target site
KDEL: Endoplasmic reticulum
KRKR: Nucleus
Hydrophobic residues: Secretion (Golgi) (Why?)
Signal sequences are not regulated, they are a property of the primary sequence

16. Role of Environment on Protein Function
In other words: Why is Regulation by Localization important?
Here’s one example:
pH changes drastically affect the ionization states of amino acid side chains
May affect ligand binding if interactions depend upon electrostatic interactions
May also cause dramatic effects in protein structure
Cathepsin D (Example #1)
Hemagglutinin (Example #2)
Diptheria toxin (Example #3)

17. Role of Environment on Protein Function #1
Cytosol
Lysosome
Lysosomal protease
pH of lysosome?
The amino terminal domain swivels due to ionization changes
Exposes active site, allows binding and charges catalytic residues

18. Role of Environment on Protein Function #2
Hemagglutinin is responsible for binding sialic acids on the host cell surface; causes RBCs to agglutinate
The amino terminus of the protein is responsible for fusion with host cell membrane
When engulfed by the cell and placed in a lysosome, the conformational change occurs

19. Role of Environment on Protein Function #3
Diptheria Toxin
Kills by inhibiting protein synthesis (A-domain)
Toxic domain released by reduction of disulfide bond inside cell
pH change causes conformational change in T-domain
T-domain punctures endosomal membrane and A-domain is released

20. 2. Regulation by Ligand Binding
Simplest method to regulate a cell is to jam something into the active site
In the multienzyme pathways of a cell, the product of one enzyme in a pathway may inhibit another
Feedback and Feed-forward Inhibition
Lots of examples available: but glycolysis is perhaps one of the best (PFK)

21. Cooperativity
Another way to fine tune enzymatic action is to jam something into another site (not the active site)
In oligomeric enzymes, the binding of a ligand may induce a conformational change that affects binding of ligand in other subunits
May be positive or negative
+=Hemoglobin
-=G3P dehydrogenase
Effector binds active site

22. Allostery
If the ligands bind to a site other than the active site, then the form of regulation is called allostery
Sequential activation
Equilibrium states

23. Post-Translational Modifications: Phosphorylation
Most common form of post-translational modification
Is Reversible
Ser, Thr and Tyr are phosphorylation targets
The large negative charge cluster of the phosphate group causes a large conformational change
Reversibility granted by having 2 specific enzyme families
Kinases: Phoshorylate proteins
Phosphatases: Dephosphorylate proteins (hydrolysis)
3 families that recognize each of the phosphorylated residues

24. Post-Translational Modifications: Phosphorylation
Effects:
Phosphoryl oxygens hydrogen bond to mainchain amide hydrogens OR
Phosphoryl oxygen forms a salt-bridge with an Arginine
Causes conformational change as residues escape the charges or are pulled/pushed as an effect of 1) or 2)
Exposes areas for other proteins to interact with

25. Post-Translational Modifications: Phosphorylation
Examples: Glycogen phosphorylase (1gpa and 1gpb)
Phosphorylation of Ser-14 leads to a major shift of the residue after rearrangement
Salt bridge formed with Arginine in loop
Active site opened

26. Post-Translational Modifications: Phosphorylation
Examples: Mitogen Activaed Kinase (MAPK)
Responsible for causing cellular changes in response to mitogen activation
Dephophorylated form
Phosphorylated form
Qi M , and Elion E A J Cell Sci 2005;118:

28. Understanding the Implications of PTMs
Key in signalling and marking proteins for termination
Mutations may block PTM sites or create them
In cancer cells, changing roles of acetylation, methylation, phosphorylation and glycosylation have major roles
In parasitic infections and bites/stings/jabs from poisonous creatures, cleaving glycosyl residues and GPI anchors helps drive the infection or accelerate death/paralysis
In the immune system, mutations in glycosylation pathways and/or glycosylation targets have dire consequences