Enzyme mechanisms

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  1. Chymotrypsin
    • digestive enz in pancreas and released to SI
    • serube protease
  2. What is diisopropylphophofluoridate (DIPF)?
    group specific reagent that modifies Ser residues
  3. What is tosyl-L-phenylalanine chloromethyl ketone (TPCK)?
    affinity label that is similar in structure to chymotrypsin substrates; covalently modifies His 57 when reacted with chymotrypsin
  4. What is N-acetyl-L-phenylalanin p-nitrophenyl ester?
    chromogenic color substrate that reacts with chymotrypsin to form p-nitrophenolate, which has a yellow color
  5. What are the two phases of chymotrypsin catalysis?
    • Burst
    • Steady state
  6. What is the burst phase?
    in chymotrypsin catalysis

    formation of acyl-enz int; concurrent with colored product formation (from what substrate?)
  7. What is the limiting step in chymotrypsin catalysis?
    deacylation step in burst phase (takes long time for enz to be "reset")
  8. What is the catalytic triad?
    • three residues involved in chymotrypsin catalysis:
    • Ser 195
    • His 57
    • Asp 102
  9. Asp 102
    • in catalytic triad of chymotrypsin catalysis
    • orients His 57 by H bonding to the imidazole ring of His 57
  10. His 57
    • in catalytic triad of chymotrypsin catalysis
    • H bonds to Ser 195
    • serves as general acid catalyst during hydrolization of peptide bond
  11. Asp 102
    • in catalytic triad of chymotrypsin catalysis
    • orients His 57
  12. What is meant by substrate specificity in relation to chymotrypsin catalysis?
    chymotrypsin only cleaves bonds on residues w/ lg, phobic side chains...due to S1 binding pocket.
  13. What is the S1 binding pocket?
    • in chymotrypsin
    • only fits side chains like Phe, Tyr, and Trp; allows binding of adj peptide into AS of chymotrypsin
  14. What is meant by P1, P2, P3, Pn?
    substrate side chain residues on N terminal side of bond to be cleaved
  15. What is meant by P1', P2', P3', Pn'?
    substrate side chain residues on carboxyl side of bond to be cleaved
  16. What is meant by S1, S2, S3, Sn?
    binding sites on protease corresponding to P1, P2, P3, Pn
  17. What is meant by S1', S2', S3', Sn'?
    binding sites on protease corresponding to P1', P2', P3', Pn'
  18. What is necessary for cleavage in the active site of chymotrypsin?
    appropriate bonding of S1 pocket to Phe, Tyr, or Trp
  19. What do trypsin, elastase, and chymotrypsin have in common?
    • serine proteases that have:
    • 40% seq idenity
    • identical mechanism
  20. What is the S1 pocket specificity for chymotrypsin?
    Phe, Tyr, Trp
  21. What is the S1 pocket specificity for trypsin?
    • Lys, Arg
    • *Arg present at bottom of pocket
  22. What is the S1 specificity of elastase?
    • Ala, Ser
    • *two Val residues allow only small side chains
  23. Subtilisin
    • bacterial protease
    • same catalytic mech as chymotrypsin but diff structure...convergent evol esempio
  24. Carboxypeptidase II
    • bacterial protease
    • same catalytic mech as chymotrypsin, but diff structure....esempio di convergent evolution
  25. What is common among ALL proteases?
    stabilization of tetrahedral TS int
  26. What are examples of cysteine proteases?
    • Papain (da papaia)
    • caspases
    • *both play roles in apoptosis (think immuno)
  27. How is the mech of cysteine proteases similar to serine proteases?
    utilizes Cys and His residues in AS just as Ser proteases use Ser and His in AS
  28. What nucleophile is created during Cys protease mech?
    S nucleophile: da His residue activating the Cys -SH
  29. What are examples of Aspartyl proteases?
    • Renin: enz in BP reg
    • Pepsin: dig enz
    • HIV protease: req for HIV prog
  30. What does two-fold symmetry mean?
    in aspartyl proteases; contain two fold symmetry that is result of two copies of a gene evolving into a single chain enz...results in pair of aspartate residues in AS
  31. What are examples of metalloproteases?
    • thermolysin: bact enz
    • carboxypeptidase A: dig enz
  32. What is unique about metalloproteases?
    contain a metal ion (usually zinc)
  33. What is the ligand for the metalloprotease metal ion?
    water molecule
  34. What are some of the characteristics of HIV proteases?
    • asp protease
    • dimer of identical subunites, each with an Asp residue
    • has flaps which close in on binding pcoket after the substrate has bound
  35. What is crixivan?
    HIV protease
  36. How does crixivan function?
    • analog of tetrahedral TS int
    • specificity to HIV protease e' da chem groups on drug that interact w/ S2, S1', S2' binding sites on HIV enz
  37. How does the HIV protease-crixivan complex work?
    • adopts conf similar to two fold symmetry of protease; AS covered by flaps
    • *two carbonyl groups on inhibitor make water mediated H bonds to an NH group in each flap (unique to HIV proteases); OH group of central alcohol is analog of negatively charged oxygen in tetrahedral TS and interacts w/ two Asp side chains at AS
Card Set
Enzyme mechanisms
MS1/Mod 1: Biochem; enzyme mechanisms
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