1. Enzyme Kinetics and Inhibition
Pratt & Cornely Ch 7

2. Enzyme Kinetics How fast an enzyme catalyzed reaction goes
Why study enzyme kinetics?
Helps us understand mechanism of enzyme (how it works)
Investigation of mutations in metabolic pathways
Understanding of regulation of biochemical reactions (up or down regulation of catalyst)

3. Simple Mechanisms Chemical mechanism Enzyme Catalyzed
How do we measure kinetics experimentally?

4. Chemical Kinetics Rate: measure product formed per second
Rate slows as reactant disappears
Measure initial rate
Do a second experiment with more starting material, and the initial rate is faster

5. Chemical Kinetics
Secondary plot: change in rate as a function of how much substrate you started with
Linear plot—does that make sense?

6. Enzyme Kinetics Complicated—two components, treated separately
First, how does [enzyme] affect rate (given large [S]?)

7. Enzyme Kinetics
Next, keep the [E] constant and low, and test how changing the [S] affects initial rates
Michaelis-Menton Treatment
[Product]
Time

8. Interpretation of Shape
Low [S]
Rate very dependent on [S]
Binding is rate limiting
High [S]
Rate independent
Saturation of E
Chemistry is rate limiting

9. Mechanism and Assumptions
E + S  ES E + P
Low [E] relative to [S]
Steady state
Initial rates
No back rxn
No pdt inhibition

10. Michaelis-Menton Kinetics
Rectangular hyperbola
Parameters
Vmax [S]
vo =
Km + [S]

11. Maximum Velocity and the Catalytic Constant
What two things contribute to the maximum velocity limit?
Amount of enzmye
Chemical ability of enzyme (catalytic constant)
Vmax = [E] kcat
Only kcat tells us about the enzyme
Maximum # of substrate molecules per active site per second
Turnover number

12. Michaelis Constant
Km is the [S] at which the reaction reaches half its maximum velocity
Physical meaning (assuming equilibrium binding): Km is the dissociation constant for ES
Km is [S] at which enzyme is half-bound
Km is measure of affinity of enzyme for S
Low Km is tight binding

13. Enzyme Efficiency
At low [S], the second order rate constant is kcat/Km
Efficient enzymes have large kcat/Km
Large kcat and/or
Small Km
Catalytic perfection at 108 or 109 M-1 S-1
Diffusion control
𝑣= 𝑘 𝑐𝑎𝑡 𝐸 [𝑆] 𝐾 𝑚 +[𝑆]
Assume large [S] and small [S]

14. Case Study: Diffusion Controlled Enzymes

15. Superoxide Dismutase: Better than Diffusion!

16. Catalytic Proficiency

17. Graphical Determination of Kinetic Parameters
Analyze hyperbola
Construct linear plot
Double reciprical

18. Non-MM Kinetics Multi-substrate Multistep reactions Allosteric enzymes
Each substrate has its own Km
Random, ordered, ping-pong
Multistep reactions
kcat not simplified to k2
Allosteric enzymes
cooperativity

19. Irreversible Enzyme Inhibition
Affinity labels
Test enzyme mechanisms
Serine protease

20. Mechanism Based Inhibitors
Suicide inhibitors
Selectivity
Targeting fast-growing cells

21. Drug Byproducts Oxidation of xenobiotics by P450 enzymes Pharmacology
Liver damage—covalent binding to cysteine

22. Reversible Inhibition Kinetics
Know types of Reversible Inhibition
Know effect on kinetic parameters
Understand why
Interpret MM plots

23. Competitive Inhibition
Added substrate can outcompete inhibitor
“Feels like…”
Same amount of Enzyme at high [S]
Needs more S to bind (lowers affinity)
Draw altered MM plot

25. Inhibition Constant for Competitive Inhibitors
vo = 𝑉𝑚𝑎𝑥[𝑆] 𝛼𝐾𝑀+[𝑆]
Alpha is the degree of inhibition
Changes the apparent KM
If KM changes from 100 nM to 300 nM, then a = 3
Depends on the concentration of inhibitor and the dissociation constant
Low Ki is better inhibitor
𝛼=1+ [𝐼] 𝐾𝑖

26. Transition State Analog
Your book presents high energy intermediate analog

27. Designing a Transition State Analog

28. Binding of Transition State Analog

29. Case Study: Orotidine Decarboxylase

30. Mechanism of Catalysis

31. Uncompetitive Inhibition
When inhibitor binds only to [ES]
Added substrate increases inhibitor effect
“Feels like…”
Less enzyme at high [S]
Enzyme has greater affinity for substrate
Draw altered MM plot

32. Noncompetitive Inhibition
Assumes simple case of inhibitor binding equally to E and ES
“Feels like…”
Less enzyme at all [S]
No effect on substrate affinity (no equil shift)
Physical explanation: inhibitor binding causes change that affects reaction, but not S binding
Very rare (nonexistent)
Draw altered MM plot

33. Mixed Inhibition Like noncompetitve, but not the simple case
Inhibitor may bind E or ES better
“Feels like…”
Less enzyme at all [S]
Overall lowering OR raising of affinity for substrate
Mixed inhibition

34. Fill in the Chart Inhibition Effect on KM Effect on Vmax
Effect on Vmax/KM
Competitive
Uncompetitive
Noncompetitive
Mixed

35. Problem 56 Use LB plot to determine parameters
[S] m M
V ( no I)
V (with I) 10
4.63 nmol/min
2.70 15
5.88
3.46 20
6.94
4.74 25
9.26
6.06 30
10.78
6.49 40
12.14
8.06 50
14.93
9.71
Use LB plot to determine parameters
What type of inhibition?
Calculate Ki.

36. Allosteric Regulation
Can be inhibition
Negative effector
Feedback inhibition
PFK regulation

37. Mechanism
PEP binding in allosteric site causes conformational shift in neighbor
An Arg essential for F6P binding is replaced with Glu
T vs. R state
Cooperative, no effect on Vmax, but only apparent KM

38. Positive Effector ADP acts with positive cooperativity
Favors R state by binding in the same allosteric site, but holding it open to lock Arg into place
Does ADP effector make sense physiologically?

39. Other Modes of Regulation
Transcriptional level
Compartmentalization
Intracellular signal
Covalent modification