Serine Protease and Its Inhibitor

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Serine Protease and Its Inhibitor

• By ???

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????????

• ??????
• 1? Orientation ????
• 2? Acid-base transfer ????
• 3? Bond Strain ????

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• ??????????????????? (concerted set)
• ?Carboxypeptidase A ,???
• ???????,???????? (sequential mechanism)
• ?Chymotrypsin ,??????

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Chymotrypsin ??????
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Chymotrypsinogen (inactive)
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Trypsin
p-Chymotrypsin (active)
R15-I16
p-Chymotrypsin
S14-R15
T147-N148
I16
L13
A149
Y146
Disulfide bonds
a-Chymotrypsin (active)
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Gly 193
His 57
Ser 195
Asp 194
Asp 102
NH3
Ile 16
?????
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Ser 195
Asp 102
His 57
Active Ser
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Chymotrypsin ??????????

• (1) Acylation
• (2) Deacylation

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Chymotrypsin ????
Acylation
Nitrophenol acetate
Kinetics of reaction
Deacylation (slow step)
CH3COOH
H2O
Two-phase reaction
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Acylation

• 1?Substrate peptide ?????
• ???????,?????? (aromatic amino acid)

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• 2?Ser-O- ?? peptide ??? carbonyl ?,??????????

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• 3?Peptide bond ??,His 57 ???? ?,?? free
  peptide N (N-????)

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• 4?Peptide C ? acylation ?? Ser 195 ?,??????

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ES
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Deacylation

• 1?????????,??? His 57

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• 2?????????? carbonyl ?,?????????,??????? His57

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• 3?Ser 195 ??????peptide,?????????

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pH?????????
pH 6
pH 7

Inactive
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pH Influences Chymotrypsin Activity
Relative Activity
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Inhibitor????????

• ?????
• TPCK (tosyl-L-phenylalaninechloromethyl ketone
  )
• DIFP (diisopropyl-fluorophosphate)
• ?DIFP ????? Ser 195 ??,?????????,??????????

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Chymotrypsin Ser195 Inhibited by DIFP
Diisopropyl-fluorophosphate (DIFP)
X
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Addition of Substrate Blocks DIFP Inhibition
No substrate
DIFP
X
Add substrate
DIFP substrate
S
Reaction time
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Inhibitor - ????

• ?????????,?????????????????????????????

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???

• 62. Chymotrypsin ???????????,???????,?????????????
  ?
• (A) Histidine 57
• (B) Aspartate 102
• (C) Serine 195
• (D) Cysteine 201

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• 78. 79.
• Chymotrypsinogen?????Chymotrypsin?
• (A) ?protein kinase?Chymotrypsinogen???
• (B) Chymotrypsinogen?cAMP??
• (C) Chymotrypsinogen??????
• (D) Chymotrypsinogen?????? (proteolysis)
• (E) ??????(dimer)??????(tetramer)

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• 83. ???Chymotrypsin????????
• (A) p-nitrophenyl acetate
• (B) Diisopropyl fluorophosphates
• (C) tosyl-L-lysine chloromethyl ketone
• (D)EDTA

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• 85. Diisopropylphosphofluoridate
  (DIPF)???chymotrypsin????,??DIPF?chymotrypsin?????
  ???????
• (A) Asp
• (B) His
• (C) Ser
• (D) Val

?????? His?Asp ?Ser