Mapping of nucleophilic centers using irreversible inhibitors

1
Basic Techniques used to determine mechanism

• Mapping of nucleophilic centers using
  irreversible inhibitors
• Stop-flow spectrophotometry
• Effect of pH on Enzyme Activity

2
Basic Techniques used to determine mechanism

• Mapping of nucleophilic centers using
  irreversible inhibitors
• Stop-flow spectrophotometry
• Effect of pH on Enzyme Activity
• Stereochemical outcomes of products
• Use of co-factors
• Metal Ion exchange
• Atom exchange

3
Same reaction Different Catalytic Mechanism
4
Another example
http//www.rcsb.org/pdb/pe/explorer/pe.htm?id1PYD
5
Beriberi

• A disease caused by a deficiency of vitamin B1
  (thiamine)
• Usually Fatal, causes mental disorders, paralysis
  and atrophy
• Seen in rice-consuming areas that practice rice
  polishing removing the thiamine rich course
  outer layers from the rice
• Seen in alcoholics (Wernicke-Korsakoff Syndrome)
  Bias the equilibrium of PDC ADH.
• Thiamin (vitamin B-1) is a water-soluble and
  heat-labile vitamin that acts as a coenzyme in
  the decarboxylation (pyruvate and ketoglutarate)
  and transketolation pathways (pentose phosphate
  pathway) of carbohydrate metabolism, and possibly
  in nerve conduction (essential for the synthesis
  of acetylcholine). http//www.emedicine.com/ped/t
  opic229.htm

6
Another example
http//www.rcsb.org/pdb/pe/explorer/pe.htm?id2OHX