Formal lab report

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Formal lab report

• Rough draft due Friday (2 copies, please)
• Any questions?
• Figures

2
Todays 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

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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

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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 gtgt I, inhibitor has no effect
• (ie. competitive inhibitor has no effect on Vmax)

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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 dont influence Km

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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

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Kinetics to determine type and potency of
inhibition

• 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

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• Non-competitive inhibitor
• Affects Vmax (moves y-intercept up)
• Doesnt affect Km (x-intercept same)

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Determining Ki

• Need to know type of inhibition
• Competitive
• Slope is increased by (1I/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

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Determining Ki

• Need to know type of inhibition
• Non-competitive Vmax in the presence of
  inhibitor Vmax
• So, determine Vmax with no inhibitor, then
  apparent Vmax values at one or more inhibitor
  concentrations

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Determining Ki

• Cant 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

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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

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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
Chymotrypsin vs. DIFP

• Doesnt look at all like a peptide bond, but
  reacts with Ser195
• Enzyme cant complete the reaction
• Active site permanently blocked