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Serine Protease and Its Inhibitor

Serine Protease and Its Inhibitor. By 第四組. 酵素基本反應機制. 三個基本動作： 1 、 Orientation 空間方向 2 、 Acid-base transfer 化學轉移 3 、 Bond Strain 構形扭曲. 以上三種方式同時發生稱為 協同式酵素催化 (concerted set) 例： Carboxypeptidase A ，羧肽酶 若先後依序發生，稱 順序式酵素催化 (sequential mechanism) 例： Chymotrypsin ，胰凝乳蛋白酶.

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Serine Protease and Its Inhibitor

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  1. Serine Protease and Its Inhibitor By 第四組

  2. 酵素基本反應機制 三個基本動作： 1、 Orientation 空間方向 2、 Acid-base transfer 化學轉移 3、 Bond Strain 構形扭曲

  3. 以上三種方式同時發生稱為協同式酵素催化 (concerted set) 例：Carboxypeptidase A ，羧肽酶 若先後依序發生，稱順序式酵素催化 (sequential mechanism) 例：Chymotrypsin ，胰凝乳蛋白酶

  4. Chymotrypsin 胰凝乳蛋白酶

  5. Chymotrypsinogen (inactive) 245 Trypsin p-Chymotrypsin (active) R15-I16 p-Chymotrypsin S14-R15 T147-N148 L13 I16 Y146 A149 Disulfide bonds a-Chymotrypsin (active)

  6. Gly 193 His 57 Ser 195 Asp 194 Asp 102 +NH3 Ile 16 催化鐵三角

  7. H O C–O–H O C–O- C = = N H–N H–O–CH2 C C H CH2 H C Ser 195 N–H N -O–CH2 C C Asp 102 H CH2 His 57 Ser 195 Asp 102 His 57 Active Ser

  8. Chymotrypsin催化步驟大約分成兩段 (1) Acylation (2) Deacylation

  9. Chymotrypsin 催化機制 O CH3–C–O– –NO2 O C O- C O-H C O CH3–C HO– –NO2 Nitrophenol Time (sec) Acylation Nitrophenol acetate Kinetics of reaction Deacylation (slow step) CH3COOH + H2O Two-phase reaction

  10. Acylation 1、Substrate peptide 進入活性區 檢查專一性位置，確定水解位置 (aromatic amino acid)

  11. 2、Ser-O-攻擊 peptide 上面的 carbonyl 碳，形成第一個過渡狀態。

  12. 3、Peptide bond 斷裂，His 57 供給氫離 子，放出 free peptide N (N-端那一半)

  13. 4、Peptide C 以 acylation 接在 Ser 195 上，成為中間產物

  14. -C-C-N-C-C-N-C-C-N- H H O- -C N- HOH O- -C N- HOH O -C N- H O -C-OH NH2- ES

  15. Deacylation 1、水分子進入活性區，結合在 His 57

  16. 2、水分子的氧攻擊上述 carbonyl 碳，形成第二個過渡狀態，並把一個氫留在 His57

  17. 3、Ser 195 放出所連結的peptide，回復原來的活化狀態

  18. pH值對酵素活性的影響 H H O C–O- = H–O–CH2 C C N–H N H–N H–N H+ C C C C H H H + Ser 195 C N–H H–N C C-H Asp 102 CH2 His 57 pH 6 pH 7 + Inactive

  19. pH Influences Chymotrypsin Activity 5 6 7 8 9 10 11 pH Relative Activity

  20. Inhibitor對酵素活性的影響 常見抑制劑： TPCK(tosyl-L-phenylalaninechloromethyl ketone ) DIFP (diisopropyl-fluorophosphate) ※DIFP 會與其上的 Ser 195 反應，然後形成固定的鍵結，使得該酵素失去活性。

  21. Chymotrypsin Ser195 Inhibited by DIFP O (CH3)2CH–O–P–O–CH(CH3)2 F = O (CH3)2CH–O–P–O–CH(CH3)2 = O-…H CH2 Ser 195 O CH2 Ser 195 Diisopropyl-fluorophosphate (DIFP) X

  22. Addition of Substrate Blocks DIFP Inhibition 100 50 0 Percent Inhibition of activity (%) No substrate + DIFP X Add substrate + DIFP & substrate S Reaction time

  23. Inhibitor - 沙林毒氣 • 沙林是一種神經毒氣，能使自主神經系統的交感神經與副交感神經立即失去平衡的毒氣。

  24. 國考題 62. Chymotrypsin 催化水解蛋白質的胜肽鍵，在催化官能基中，下列何者參與親核性催化機制？ (A) Histidine 57 (B) Aspartate 102 (C) Serine 195 (D) Cysteine 201

  25. 78. 79. Chymotrypsinogen如何轉換成Chymotrypsin？ (A) 由protein kinase將Chymotrypsinogen磷酸化 (B) Chymotrypsinogen與cAMP結合 (C) Chymotrypsinogen與鈣離子結合 (D) Chymotrypsinogen經由蛋白水解 (proteolysis) (E) 由原本二聚體(dimer)聚合為四聚体(tetramer)

  26. 83. 何者是Chymotrypsin之非可逆性抑制？ (A) p-nitrophenyl acetate (B) Diisopropyl fluorophosphates (C) tosyl-L-lysine chloromethyl ketone (D)EDTA

  27. 85. Diisopropylphosphofluoridate (DIPF)為酵素chymotrypsin之抑制劑，是因DIPF與chymotrypsin內核者胺基酸做共價結合？ (A) Asp (B) His (C) Ser (D) Val ※催化鐵三角： His、Asp、Ser

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