Lecture 9 Web: pollev.com/ucibio Text: To: 37607 Type in: 169964 <your question>

MS-MS to sequence proteins

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

MS Example

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

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

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

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

Measuring Initial Velocity at different [S]

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

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

The Michaelis-Menten kinetics graph

How to estimate VMax? y = mx + c

The Lineweaver-Burk plot

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