Download - Chapter 6 Protein Function: Enzymes Part 2
Chapter 6
Protein Function: Enzymes
Part 2
Enzymes, Part 2
– Chemical mechanisms of catalysis: Chymotrypsin– Multi-substrate enzyme reaction kinetics– Reversible enzyme inhibitors and inhibition kinetics
More fun with Practicase– Regulation of enzyme activity.
Learning Goals: To Know
Part of Worked Example 6.1An enzyme, happyase, is discovered to catalyze:
SAD HAPPY
It is known that the kcat is 600 s-1.
When [Et] = 20 nM, and [SAD] = 40 μM, the reaction velocity (vo) is 9.6 μM/s. What is the KM?
Solution combines the kcat equation and the Michaelis-Menten equation.
kcat = Vmax/ [Et] so: Vmax = kcat [Et]
Substitute into Michaelis-Menten equation for Vmax:
vo = [ Vmax (S)] / [Km + (S)]
vo = [kcat [Et] (S)] / [Km + (S)] Answer: Km = 10μM
So Far… … … it has just been single substrate:
S + E ES E + P
Now lets go to multiple substrates and multiple products (we will only go as much as 2 substrates 2 products):
Enz
A + B C + D
Multiple Substrate Reactions
Lineweaver Burke Plot – Enzymes forming Ternary Complexes – Ordered or Random
Lineweaver Burke Plot – Enzymes Without Ternary Complexes
Enzyme Inhibition
Inhibitors are compounds that decrease enzyme’s activity
•Irreversible inhibitors (inactivators) react with the enzyme• One inhibitor molecule can permanently shut off one enzyme molecule• They are often powerful toxins but also may be used as drugs
•Reversible inhibitors bind to and can dissociate from the enzyme• They are often structural analogs of substrates or products• They are often used as drugs to slow down a specific enzyme
•Reversible inhibitor can bind: • to the free enzyme and prevent the binding of the substrate• to the enzyme-substrate complex and prevent the reaction
Reversible Inhibitors - Competitive
Lineweaver Burke - Competitive Inhibition
α = 1 + [I]/Ki
-1/Km
-1/αKm
Reversible Inhibitors - Uncompetitive
Lineweaver Burke: Uncompetitive Inhibition
α’ = 1 + [I]/Ki’
Reversible Inhibitors – Mixed Inhibition
Lineweaver Burke – Mixed Inhibition
α = 1 + [I]/Ki
α’ = 1 + [I]/Ki’
α’/Vmax
- α’/αKm- 1/Km
Shows Region of Inhibitor Effect
Apparent Vmax or Apparent Km refers to y or x axis intercept only. The Next Slide is MUCH BETTER
Calculation of Enzyme Constants
Type of Inhibition X axisintercept Y axis intercept
None -1/Km 1/Vmax
Competitive -1/αKm 1/Vmax
Uncompetitive - α’/Km α’/Vmax
Mixed - α’/αKm α’/Vmax
EOC Problem 12: Lets figure out what sort of inhibitor ibuprofen (active ingredient in Advil) is. Ouch!!! or Ahhhh!
And, next there is our friend Practicase and inhibitors.
Inhibition of Practicase
[Studentose],mM vo, uninhibited vo Inhbitor A vo Inhibitor B vo Inhibitor C
1 12 4.3 5.5 5
2 20 8 9 8.69
4 29 14 13 13.7
8 35 21 16 19.6
12 40 26 18 22.2
Inhibitor A at 1 mM
Inhibitor B at 3 mM
Inhibitor C at 50 μM
L-B plots of Practicase Inhibitiors
Calculation Inhibitor A’s Practicase Ki :
Be sure to calculate the Ki’s for the other inhibitor.
Is the inhibitor a potential drug? Compare the Ki to the KM…what does this tell you?
We already know KM = 3.33 mM and Vmax= 50 µmoles/mL/s and it is a Competitive Inhibitor
The Inhibited curve intersects the X-axis at -0.1 mM, thus
-1/αKM = -0.1 mM
Solving for α, α = 3
So, α = 1 + [ I ]/Ki we know the inhibitor in the experiment was 1 mM
Thus, 3 = 1 + 1mM/Ki
2 = 1mM/Ki
Ki = 0.5 mM
Thus inhibitor A binds the enzyme BETTER than the substrate!
Practicase Inhbitiors
Calculation of Mixed Inhibitor’s Ki’sThis is Inhibitor 2
This inhibitor has α and α’… to calculate Ki and Ki’
So, FIRST you need to calculate α’ … the best place to do that is from the y-axis intercept = α’/Vmax
Then to get α, go to the x-axis intercept = α’/αKm
then from each, α and α’ you can determine Ki and Ki’
Remember this one inhibitor binds both to E and ES.
Enzymes and Fashion
“Stonewashed Jeans”
Jeans are washed with cellulase (an enzyme that hydrolyzes celluose – major component in cotton) at a low concentration for a short time…..the effect looks “stonewashed”.
If they were really stonewashed how would they get all the stones out of the pockets?
Irreversible Inhibition
Mechanism Based
Suicide Inhibitors
Effect of pH on Enzyme Activity
Effect of pH on Chymotrypsin
Chymotrypsin – Our Model Enzyme
Active Site of Chymotrypsin with Substrate
Aromatic Part of Substrate = Green
Chymotrypsin – Our Model Enzyme
Amide Nitrogens Stabilize Oxyanion
Reactive Groups in Enzymes are Either:
Chymotrypsin Mechanism Step 1: Substrate Binding
Chymotrypsin MechanismStep 2: Nucleophilic Attack
Chymotrypsin Mechanism
Step 3: Substrate Cleavage
Chymotrypsin MechanismStep 4: Water Comes In
Chymotrypsin MechanismStep 5: Water Attacks
Chymotrypsin Mechanism Step 6: Break-off from the Enzyme
Chymotrypsin Mechanism Step 7: Product Dissociates
Drug Company Recruiting Ad
The importance of structural protein chemistry !!! And transition state analogs…
Which bind the active site exceptionally well.
from C&EN, Aug 13, 2007
Hexokinase Reaction : Induced Fit
What happens when glucose binds
Induced Fit with Glucose Binding
Daniel Koshland 60’s
Remember in Part 1 of this Chapter glucose prevented thermal destruction of hexokinase…EOC problem 4.
Xylose is One Carbon Shorter than Glucose
Xylose causes Hexokinase to become an ATPase
When Xylose reacts with Hexokinase – it causes induced fit and Mg++ ATP binds…
but xylose does not exclude water from the active site where the 6th carbon would be.
Normally the Induced fit is the active form, and catalyses the phospho-transfer from ATP to glucose glucose-6-phosphate + ADP, but when xylose is there:
Xylose + H2O + ATP Xylose + ADP + Pi…. A futile use of ATP!
Enzyme activity can be regulated
• Regulation can be:– noncovalent modification– covalent modification
– and either• irreversible• reversible
Noncovalent Modification: Allosteric Regulators
The kinetics of allosteric regulators differ from Michaelis-Menten kinetics.
Allosteric Effectors – Bind to Allosteric Site
Feedback Inhibition is the Classic Form of Allosteric
Inhibition
Allosteric Enzymes Often Have Sigmoid Kinetics
Allosteric Positive and Negative Regulators: Affecting KM
Allosteric Positive and Negative Regulators: Affecting the Vmax
Enzyme Regulation by Covalent Modification
Glycogen Synthase Regulation: Both Allosteric and Covalent
From Ch 15
PP1: Protein Phosphatase-1
Zymogen Regulation
Things to Know and Do Before Class
1. Know the chymotrypsin reaction and the concept of how enzymes participate in the reaction.
2. Kinetics of multisubstrate reactions.
3. Types and kinetics of reversible enzyme inhibitors. And, the importance of Ki.
4. Types enzyme regulation and their Michaelis Menten kinetics.
5. Be able to do EOC Problems 12, 18, 19.