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Formal lab report

Formal lab report. Rough draft due Friday (2 copies, please) ¿ Any questions? Figures. Today’s lab. Measure V o vs. [S] for fumarate (fwd rxn) or malate (rvs rxn) For everyone: +/- citrate (reversible inhibitor) Fumarate: you want to start ~ same absorbance

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Formal lab report

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  1. Formal lab report • Rough draft due Friday (2 copies, please) • ¿Any questions? • Figures

  2. Today’s lab • Measure Vo vs. [S] for fumarate (fwd rxn) or malate (rvs rxn) • For everyone: +/- citrate (reversible inhibitor) • Fumarate: you want to start ~ same absorbance • Molar absorptivity depends on wavelength • Higher initial concentrations: use wavelength where fumarate absorbs more weakly • Malate: measure at 240nm

  3. Kinetics & Reversible Inhibition • Determine mechanism of inhibition • Competitive (alter binding, raise apparent Km) • Non-competitive (alter catalysis, lower apparent Vmax) • Determine potency of inhibition • Ki: “Inhibition constant” • Lower Ki means more potent inhibitor

  4. Mechanism of inhibition • Competitive: • Inhibitor competes with substrate for binding to the active site • Inhibitor binds FREE ENZYME • Inhibitor must “look like” substrate • Note: when [S] >> [I], inhibitor has no effect (ie. competitive inhibitor has no effect on Vmax)

  5. Mechanism of inhibition • Competitive: • Inhibition constant Ki EI ↔ E + I • Lower Ki = stronger affinity ie. more potent inhibitor (inhibits at lower concentrations)

  6. Mechanism of inhibition • Mixed (eg. Non-competitive) • Inhibitor binds somewhere else (not active site) • Bind either E or ES (to form EI or ESI complexes) • Mixed alter both Km and Vmax • Pure non-comp alter Vmax, but don’t influence Km

  7. Mechanism of inhibition • Mixed (eg. Non-competitive) • Since “Ki” describes two potential equilibria, harder to get a handle on • But still, lower Ki ~ stronger inhibitor

  8. Lineweaver-Burk plot Y-intercept = 1/Vmax X-intercept = -1/Km Competitive inhibitor affects Km, not Vmax L-B plot: same y-intercept, x-intercept moves to the right Kinetics to determine type and potency of inhibition

  9. Non-competitive inhibitor Affects Vmax (moves y-intercept up) Doesn’t affect Km (x-intercept same)

  10. Determining Ki • Need to know type of inhibition • Competitive: • Slope is increased by (1+[I]/Ki) • So, make L-B plot at 0 inhibitor and compare its slope to the slope at one or more inhibitor concentrations to determine Ki

  11. Determining Ki • Need to know type of inhibition • Non-competitive: Vmax in the presence of inhibitor ~ V‘max • So, determine Vmax with no inhibitor, then apparent Vmax values at one or more inhibitor concentrations

  12. Determining Ki • Can’t directly compare Ki values for different types of inhibitors, but gives you some idea of their potency (~ Kd) • Need to know type of inhibition before calculating Ki

  13. Irreversible inhibitors • Potential for higher effectiveness • Permanently inhibit an enzyme or take it completely out of commission • Drug discovery • “Suicide inhibitors” • Also takes the inhibitor out of commission

  14. Irreversible inhibitors • Mechanism-based inhibitor • Acts like a substrate during initial parts of the reaction • Often (always) forms covalent bond with enzyme • Insensitive to later steps • “Stalls” before release • Good for studying enzyme mechanisms

  15. Doesn’t look at all like a peptide bond, but reacts with Ser195 Enzyme can’t complete the reaction Active site permanently blocked Chymotrypsin vs. DIFP

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