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

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

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

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

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

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

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

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?

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?

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

Experimental rationale

Degradation of proteins Unfolding Ubiquitination Chaperones

Proteosomal degradation of proteins

Causing protein degradation: Theory Hydorophobic moiety

New technique What are the most important things to establish?

Causing protein degradation: Theory

Causing protein degradation: Practice

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

Luciferase activity?

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

HA Tag degradation alone?

Kinetics

Ub-Proteosome?

Membrane proteins?

Whole animals?

Does it do anything fun?

Phenotypic readouts?

Phenotypic readouts in animals?

New technique What are the most important things to establish?