Enzyme Concepts/Kinetics
How does an enzyme increase the rate of a reaction?
An enzyme increases the rate of a reaction by lowering the activation energy barrier, thus increasing the number of molecules that can reach the transition state.
Bonus: What are the 4 strategies an enzyme can use to lower the activation energy barrier?
Where is Chymotrypsin synthesized, and how it is activated?
Chymotrypsin is synthesized in the pancreas. It is activated when Trypsin cleaves between R15-I16 residues.
Bonus: What part of I16 is involved in forming a salt bridge and what residue does it form that salt bridge with?
How do Reversible Inhibitors bind to enzymes? How do Irreversible Inhibitors bind to enzymes?
Reversible Inhibitors bind through noncovalent interaction to allow the inhibitor to dissociate from the enzyme
Irreversible Inhibitors bind through covalent interactions
Bonus: What does a low Ki indicate?
What is Cooperativity? What does it correspond to?
Cooperativity is when binding at one active site impacts the binding at a different active site
It corresponds to the shape of the binding curve
Bonus: What other term is used to refer to cooperativity?
What are the 4 components of a phospholipid?
Name 2 differences between Phosphoglycerides and Sphingomyelin.
Fatty Acid Tail, Platform, Phosphate Group, & Alchol
Phosphoglycerides contain ester linkages and Sphingomyelin contains amide linkages
Sphingomyelin contains a Spingosine tail, while Phosphoglycerides do not
Bonus: Name the 2 types of Glycolipids.
Describe the 3 Chemical Complementarity Models.
Lock & Key: Enzyme & substrate fit together like a lock & key without any conformational change
Induced Fit: Enzyme undergoes conformational change after substrate binds
Conformational Selection: Enzyme undergoes conformation change before substrate binds
Bonus: Which one of these models incorporates a "lock & key" type interaction?
Describe the 2 Catalytic strategies Chymotrypsin uses.
1. Covalent Catalysis: It forms a covalent bond with the substrate which is stabilized by the oxyanion hole
2. Acid/Base Catalysis: His 57 accepts a proton from Ser195
Bonus: What 3 residues does Chymotrypsin cleave after? What's the 4th one that has a lower affinity?
What is Uncompetitive Inhibition? How is Km and Vmax impacted?
Draw the corresponding Lineweaver-Burk Plot.
An uncompetitive inhibitor binds to the ES complex
It lowers Vmax and Km
Bonus: You have 1 minute to Google and describe an example of an uncompetitive inhibitor.
What is an Allosteric Effector? What does is affect? Provide 2 examples of an Allosteric Effector that relates to ATCase.
An allosteric effector is when binding to a regulatory site impacts binding to an active site.
It effects the position of the curve
CTP and ATP are allosteric effectors of ATCase
Bonus: How does CTP and ATP shift the ATCase binding curve?
Describe the Liquid Disordered Phase and the Gel Phase.
Liquid Disordered = Fatty acid tails highly dynamic, van der waals interactions reduced, rapid lateral diffusion (high fluidity)
Gel = Fatty acids are extended, maximal van der waals interactions, low motion & lateral diffusion (high rigidity)
Bonus: How many fatty acid tails would a lipid forming a micelle contain?
Describe the 3 assumptions for the Michaelis-Menten Model.
1. Initial Velocity: Recording the rate at the beginning of the reaction gets rid of k-2
2. Catalytic Rate: k2 is the slow step, and therefore the rate-determining step
3. Steady State: Rate of formation = Rate of breakdown
Bonus: What are the equations for Km, Vmax and Catalytic Efficiency?
What 3 residues are involved in the formation of the oxyanion hole? What is the purpose of the oxyanion hole?
Gly193, Asp194, Ser195
The purpose is for transition state stabilization and to optimize substrate binding.
Bonus: What intermediate does the oxyanion hole stabilize?
What is a Noncompetitive Inhibitor? What does it do to Vmax and Km?
Draw the corresponding Lineweaver-Burk Plot
A noncompetitive inhibitor binds to the ES or free enzyme
It lowers Vmax, but has no impact on Km
Bonus: You have 1 minute to Google and describe and example of a noncompetitive inhibitor.
Describe the structure of ATCase. How was this structure mapped out?
ATCase is a dodecameric enzyme with 6 catalytic subunits and 6 regulatory subunits.
The active sites were determined through the use a molecule called PALA
Bonus: How did PALA work?
A yeast cell residing in a dog's ear is shaken out into the cool air.
What are two ways that a cell can adapt its membranes to withstand that change in temperature?
The yeast cell is going from warm -> cold therefore it needs to increase membrane fluidity
It needs to decrease the number of carbons in its fatty acid tails and it needs to increase the number of double bonds in its fatty acid tails
Bonus: What will this increase in membrane fluidity do to the Transition Midpoint (Tm)?
How will a [Product] < [Substrate] contribute to ΔG and ΔG°'?
ΔG = It will contribute favorably
ΔG°' = It will contribute unfavorably
Bonus: Will a reaction be spontaneous if it has a Keq > [products]/[substrates]?
What residues are involved in the Catalytic Triad? How do these residues impact one another to start the Chymotrypsin reaction?
Asp102: Makes His57 a better base by positioning it correctly with hydrogen bonding & increasing its pKa through electrostatic effects
His57: Acts a general base to activate Ser195 as a nucleophile
Ser195: Activated as a nucleophile
Bonus: What type of multiple substrate reaction is Chymotrypsin? What are the other 2 multiple substrate reactions talked about in class?
What is a Competitive Inhibitor? What does it do to Vmax and Km?
Draw the corresponding Lineweaver-Burk Plot.
A competitive inhibitor binds to free enzyme
It raises Km, but has no impact on Vmax
Bonus: Describe 2 examples of a competitive inhibitor that Dr. Wright talked about in lecture.
What two states does ATCase exist in?
Aspartate binds to ATCase. What state does it exist in then? What cooperativity model describes this change?
ATCase exists in the Relaxed (R) and Tense (T) state
When Aspartate binds to ATCase, all of the active sites enter into the R state because ATCase follows the Concerted Model.
Bonus: How does ATP impact T -> R change? How would ATP impact the L value?
Describe a Lipid Raft. How is it efficient?
Localized areas of low membrane fluidity that keep proteins that function together near each other. It increases the efficiency of signal transduction by keeping important proteins and receptors together.
Bonus: How does cholesterol impact membrane fluidity?
Given the following graph (I will draw), what is:
1. Vmax
2. kcat
3. Km
4. Catalytic Efficiency
1. 10
2. 333
3. 2
4. 166 or 167 (depending on how you round)
Bonus: Which of these values would you use to compare data with another lab and why?
Place the following steps in order.
1. Amino Product Leaves
2. Collapse of tetrahedral intermediate to break bond between enzyme and substrate
3. Water binds
4. Nucleophilic attack on carbonyl carbon of peptide bond to form tetrahedral intermediate
5. Carboxyl product leaves
6. Nucleophilic attack on carbonyl carbon of acyl-enzyme forms tetrahedral intermediate
7. Protein binds
8. Collapse of tetrahedral intermediate to break peptide bond
1. Protein binds
2. Nucleophilic attack on carbonyl carbon of peptide bond to form tetrahedral intermediate
3. Collapse of tetrahedral intermediate to break peptide bond
4. Amino Product Leaves
5. Water binds
6. Nucleophilic attack on carbonyl carbon of acyl-enzyme forms tetrahedral intermediate
7. Collapse of tetrahedral intermediate to break bond between enzyme and substrate
8. Carboxyl product leaves
Bonus: Elastase has a P1 binding pocket specific for small nonpolar residues. Describe Elastase's binding pocket.
Given the following graph, determine:
1. Km
2. Vmax
3. kcat
4. Catalytic Efficiency
1. 0.5
2. 0.5
3. 16 or 17 (depending on how you round)
4. 32 or 34 (depending on how you round)
Bonus: What is the slope?
Describe how an Allosteric Enzyme can be "turned on or off".
An allosteric enzyme can be "turned on or off" by shifting the binding curve. The sigmoidal binding curve associated with allosteric enzymes has large responses in activity when there are small changes in [substrate] near K0.5. The farther the
Bonus: What happens to K0.5 and kcat when ATP binds to ATCase?
Na+ is being pumped against its electrochemical gradient out of the cell.
Do you expect ΔG to be positive or negative?
Do you expect ΔV to be positive or negative?
What type of transport will be used and what protein will mediate this transport?
ΔG will be positive because Na+ is going against both its electrical and chemical gradients
ΔV will be positive because Na+ is being pumped out of the cell
Active transport mediated by pumps
Bonus: What would the case be for Cl- being pumped down its electrochemical gradient into the cell?