1. Lecture 9 Web: pollev.com/ucibio Text: To: 37607
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2. MS-MS to sequence proteins

3. Random fragmentation A-B-C-D A-B-C-D A-B-C-D A-B-C-D A- B-C-D A-B- C-D
Signal intensity
m/z
A-B-C
A-B-C-
A-B- A
A-B-C-D
A-B-C-D

4. MS Example

5. Using MS to generate a “proteome”
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6. Using MS to generate a “proteome”
Copyright  2013 Pearson Canada Inc.

7. So, enzymes have active sites.
Looked at enzyme structure = function
Do we need to also study “kinetics?”

8. Simplifying kinetics Initial rate = [P] ≈ 0 Practically 100% ES  P
At GIVEN [E], rate ∝ on [S]

9. Measuring Initial Velocity at different [S]

10. Michaelis-Menten kinetics: A model
Makes several assumptions:
1. Non-equilibrium (Vo)
2. At given [E]
3. [S] ≫ [E]
4. At steady state:
ES formation=ES breakdown

11. Determining Km V 0 = V Max [S] S + K m S + K m = V Max [S] V 0
S + K m = V Max [S] 0.5 V Max
S + K m = 2[S]
K m = [S] when V o = ½ V Max

12. The Michaelis-Menten kinetics graph

13. How to estimate VMax?
y = mx + c

14. The Lineweaver-Burk plot

15. The turnover number (k2 or kcat)
Turnover number = number of S molecules  P per second