More about enzymes and kinetics 917

1
More about enzymes and kinetics 9/17

• More about M-M kinetics hyperbolic curves, and
  approximations
• LB plots, straight lines, and exact predictions
• Why is it valuable to know km, Vm, V1/2, etc with
  respect to cell culture or drug metabolism by
  cells?
• Drugs as non-competitive and competitive
  inhibitors in cells
• LAST MATERIAL FOR FRIDAYS EXAM
• 9/19-50 points (bring scantron)
• Exam will be Ch 1-3(mostly review) and 4-6
• Exam will emphasize what is in the notes
• Textbook obviously should help understand the
  notes and is important, especially the problems
  in the back of the chapters.

2
The active site is where the substrate binds to
the enzyme. What are the six features of an
active site?

• 1) AS is small part of total!
• 2) AS spatial in 3-D!
• 3) AS hold substrate by using its specific
  charges on its amino acids to attract substrate!
• Ionic, Hydrogen, Van der Waals Forces
• Covalent bonds only at transition state!
• 4) AS located in peptide cleft!
• 5) Substrate/Cleft perfectly align!
• 6) Substrate/Enzyme binding causes induced fit at
  site!
• Induced fit result in change to shape of entire
  enzyme!

3
Competitive and Non-competitive Inhibitors bind
to different sites!
4
Competitive inhibitors change Km!-MMBecause
fewer sites are available to the
substrate!Non-Competitive Inhibitors change
Vm!-MM Because fewer enzymes are available to
the substrate!
5
Competitive inhibitors change 1/Km!-LBBecause
fewer sites are available to the
substrate!Non-Competitive Inhibitors change
1/Vm!-LB Because fewer enzymes are available to
the substrate!
6
LB Plot with inhibitorsIs A or B competitive and
non-competitive?
7
Irreversible Enzyme Inhibitors DFP is similar to
the Sarin Nerve Gas for use on humans in warfare
8
Consider these problems1) With respect to 1/Km
and 1/Vmax, which of these two Lineweaver-Burke
Plots (See below) is from a system experiencing
Competitive and Non-Competitive Inhibition? 2)
If you need the product of a reaction that is
irreversibly inhibited (I.e. Sarin Nerve Gas
effect on acetylcholinesterase druign warefare),
what must the cells of your body do to survive in
the presence of the irreversible inhibitor?
9
In this example from Dr. Wilsons research
cranberry juice stimulates an enzyme (nitric
oxide synthase) in the endothelial cells of
arteries to cleave nitric oxide (NO) off of the
amino acid arginine. NO then causes smooth
muscle cells to relax or dilate, which opens-up
blood vessels in the heart (in theory) resulting
in improved blood flow and a remission of painful
angina.

• The system consists of a piece of rat aortic
  artery in a bath of saline exposed to one of
  several chemicals. Contraction is measured as
  increased grams tension (Y-axis) caused by the
  contraction/relaxation(dilation) of smooth muscle
  in the tissue. Endothelial cells were destroyed
  by rubbing the inside of the vessel.
• 3) The endothelium is a layer of cells that line
  a blood vessel. Where is the enzyme located?B)
  L-NAME (L-n-monomethyl-arginine) blocks this
  effect, but an overdose of arginine causes a
  return of the effect. What type of inhibition
  does L-NAME exert on NOS?
• 4) What would the LB Plot look like for the
  effect of L-NAME on the formation of NO?
• 5) Would 1/Km or 1/Vmax have changed when L-NAME
  was present?
• PE phenyephrine, looks like epinephrine and
  contracts smooth muscle
• ACHacetylcholine-dilates smooth muscle in vessel
  by stimulating NOS
• CBJCranberry juice applied to cells of system
• ArgThe amino acid arginine, the substrate from
  which NO is produced
• L-NAME a drug that makes vessels contract by
  interacting with NOS

10
Figure From Maher MA, Mataczynski H, Stefaniak
HM, Wilson T. Cranberry juice induces nitric
oxide-dependent vasodilation in vitro and in vivo
and its infusion transiently reduces blood
pressure in anaesthetized rats. J Med Foods
20003141-147.