Multisubstrate reaction

• When is the michaelis-Menten model not valid?

  If two substrates can vary in conc
• Why can't multiple substrate not use michaelis menten?

  each substrate has its own Km
• what is Km?

  how much substrate (conc) is needed to reach half of Vmax
• How to use Michaelis-Menten for two substrate?

  Have one substrate fixed at high concentration (mass excess)
• Why use lineweaver-Burk?


  To determines apparents Km
• What are some reaction mechanism?

  Both substrate bond at once

  Substrate binds and leave sequentially
• what is a ternary complex?

  3 molecules bound together
• When does ternary complex form?


  when enzyme binds to both substrates at the same time
• What is an example of sequential displacement reaction?

  Lactate dehydrogenase
• what is a ping-pong mechanism?

  Double displacement
• what is double displacement?

  E binds to S1 first does something before second substrate. ES1 binds to second substrate
• Sequential displacement reaction

  
• Double displacement reaction

  
• Sequenital displacement reaction:

  Lineweaver-Burk plot similar to mixed inhibition, but:

    - Increasing S2 decreases slope

    - Apparent Vmax increases & KM decreases
• Double displacement reaction:

  Lineweaver-Burk plot similar to uncompetitive inhibition:

    - Increasing S2 decreases intercepts

    - Apparent Vmax & KM increase
• What does the Km increase in a double displacement mean?

  Km does not imply it has a weaker affinity (allosteric factors)
• What is chymotrypsin?


  Proteolytic enzyme in animals
• What does chymotrypsin do?


  Cleave peptide bonds on C-terminal side of Large hydrophobic aa (Trp, Tyr, Phe, Met)
• what mechnanism does chymotrypsin use?

  double displacement
• How can you tell which residues are involved in catalysis?

  Use irreversible inhibitors to identify catalytic residues.
• Why use irreversible inhibitors?

  Bind to active site
• What is used to map active site of chymotrypsin?


  DIPF (diisopropyl phosphofluoridate) - OH group specific reagent
• Why use DIPF?


  only binds to serine 195
• Which amino acids lie in the cleft in the surface of the enzyme and joined by hydrogen bondAsp102, Ser195?

  Ser195, His57
• peptide Hydrolysis by chymotrypsin:

  1. Substrate binding, proton abstraction
  2. Unstable tetrahedral transition state
  3. Acyl enzyme intermediate, release of first peptide
  4. Abstraction of proton from water by His57
  5. Second tetrahedral transition state
  6. Regeneration of active site, release of second peptide
• Substrate binds:

  Through R1 hydrophobic interactions. Protons abstraction from Ser195 forms alkoxide ion
• how is the tetrahedral transition state achieved? (chymotrypsin)

  Alkoxide ion attacks peptide carbonyl group
• what holds the tetrahedral intermediates in place?

  oxyanion hole
• What do proteolytic enzymes use?

  a catalytic triad
• Examples of evolution catalytic triad:

  • Chymotrypsin (tryspin, elastase)
  • Subtilisin-like proteases
  • Wheat carboxypeptidase II
• Why serine 195?


  most reactive of 27 serine in chymotrypsin
• What forms the chymotrypsin catalytic triad?

  His 57, Asp102 and Ser195
• How does His57 increase reactivity of ser195?

  polarises the OH group of ser195, So ser195 is acts a general base catalyst due to the highly reactive alkoxide ion
• catalytic triad in subtilisin:

  mutated S221, H64 and D32. Each to alanine
• What are chymotrypsin, trypsin and elastase?

  Serine-proteases
• chymotryspin cleaves...


  after amino acids with large hydrophobic side chains
• trypsin cleaves...


  after amino acids with long, positively charged side chains
• elastase cleaves ...


  after amino acids with small side chain (Ala, ser)