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Last class Enzyme kinetics – MM equation V0, Vmax, Km MM vs LWB.

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Presentation on theme: "Last class Enzyme kinetics – MM equation V0, Vmax, Km MM vs LWB."— Presentation transcript:

1 Last class Enzyme kinetics – MM equation V0, Vmax, Km MM vs LWB

2 Inhibitors What is most important part of enzyme?
What are major sites of inhibitor binding?

3 Inhibitors: Competitive
E + S ES E + P +I KiEI EI Kmapparent ? Vmaxapparent ?

4 Inhibitors: Non-competitive
E + S ES E + P +I +I KiEI KiESI EI ESI KiEI = KiESI  Kmapparent ? KiEI = KiESI  Vmaxapparent ?

5 Inhibitors 1/vo 1/[S] Which line is WT, which one is +I?
What kind of inhibition?

6 Percent inhibition How much is a reaction inhibited at a given [I]
1 − Slope or rate WITH I Slope or rate WITHOUT I x 100 ↑ [I]  ? % inhibition? ↑ [I]  ? Ki

7 Percent inhibition How much is a reaction inhibited at a given [I]
1 − Slope or rate WITH I Slope or rate WITHOUT I x 100 ↑ [S]  ? % inhibition?

8 Lab Fill out tables correctly! Determine [I]50
Measure %inhibition at different [S] – Why? Plot kinetics at different [S] w/ and w/o I – Why?

9 Regulating protein activity – Paper 2
What was main purpose/hypothesis? Approach?

10 Experimental rationale

11 Degradation of proteins
Unfolding Ubiquitination Chaperones

12 Proteosomal degradation of proteins

13 Causing protein degradation: Theory
Hydorophobic moiety

14 New technique What are the most important things to establish?

15 Causing protein degradation: Theory

16 Causing protein degradation: Practice

17 Luciferase ↓ Luciferase activity = ↓ Luciferase protein? So now what?

18 Luciferase activity?

19 HA tag VS GFP ↓ HA = ↓ Protein? So now what?

20 HA Tag degradation alone?

21 Kinetics

22 Ub-Proteosome?

23 Membrane proteins?

24 Whole animals?

25 Does it do anything fun?

26 Phenotypic readouts?

27 Phenotypic readouts in animals?

28 New technique What are the most important things to establish?

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