Download Enzymes: Principles of Catalysis

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
Drug Design Example: HIV Protease
Transition State Analogs
Example Acid-Base Catalysis: RNase A
Enzyme responsible
for degrading RNA in
the cell
Example AcidBase Catalysis:
RNase A
Example Acid-Base Catalysis: RNase A
1) The different protonation states of two active site histidines
(His12 and His199) (See PDB Entry 4AO1)
His119 pKA=6.03
His12 pKA=4.72
Example Acid-Base Catalysis: RNase A
His12: Expected to be fully deprotonated
His119: Expected to be partially protonated
His119 pKA=6.03
His12 pKA=4.72
Example Acid-Base Catalysis: RNase A
So how does this difference in pKA arise?
Example Acid-Base Catalysis: RNase A
2) 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?
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
Protein Regulation
There are three basic mechanisms to control/regulate
protein function
1. Localization of the protein and/or its partners
2. Binding of effector molecules or Posttranslational
Modifications
3. Physical amounts of protein and the lifetime of each
molecule
• This is the simplest mechanism to control protein
function
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
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.
2. Regulation by Effector Molecules –
Covalent Modification
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
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
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)
Role of Environment on Protein Function #1
Cytosol
Lysosomal protease
pH of lysosome?
The amino terminal domain swivels
due to ionization changes
Exposes active site, allows binding
and charges catalytic residues
Lysosome
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
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
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)
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
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
Post-Translational Modifications: Phosphorylation
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
Post-Translational Modifications: Phosphorylation
Effects:
1) Phosphoryl oxygens hydrogen bond to mainchain amide
hydrogens
OR
2) Phosphoryl oxygen forms a salt-bridge with an Arginine
3) Causes conformational change as residues escape the
charges or are pulled/pushed as an effect of 1) or 2)
4) Exposes areas for other proteins to interact with
Post-Translational Modifications: Phosphorylation
Examples: Glycogen phosphorylase (1gpa and 1gpb)
1) Phosphorylation of Ser-14 leads to a major shift of the
residue after rearrangement
• Salt bridge formed with Arginine in loop
• Active site opened
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;118:3569-3572
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