1. Zymogen/proenzyme (inactive enzyme precursor)
Feed forward reaction (positive feedback)
Ser (covalent bond) His (general acid/base) Asp (orient His)
Serine protease
Catalytic triad (Ser His Asp)
Substrate-binding pockets (selectivity)
Typical enzymes
Mechanism Detail
Transition-State Stabilization
Two-lobed structure
Aspartic protease
Mechanism (LBHB catalysis)
Ex : HIV-1 protease
Inhibitor drugs
Intramolecular rearrangement
Chorismate Mutase
Via chair Mechanism and Transition State Analog (TSA)
Near-Attack complex (NAC)

2. Ch14 Part III 重點整理 What Can Be Learned From Typical Enzyme Mechanisms?
Part III用三種酵素的例子來描述前面提過的mechanisms:
例二: The aspartic protease:
→general acid-base
catalysis,
LBHB
例三: Chorismate mutase:
→ NAC
例一: The serine protease:
→ covalent catalysis,
general acid-base catalysis,
substrate selectivity,
LBHB

3. The serine protease: (以Ser為活性中心的蛋白酶)
Serine protease family 包含:
-消化道酵素:typsin, chymotrypsin, elastase
(分泌時以zymogens型態出現)
- 凝血蛋白酶:Thrombin (詳見Ch15)
Catalytic Triad (三個主要的活性中心)
His57, Asp102, Ser195
Zymogen
(酵素前驅物)
Active enzyme
經水解反應 (hydrolysis)
或構形改變 (configuration change)
使active site 暴露
*Positive feedback
可自身活化

4. Covalent catalysis & general acid-base catalysis
詳見: “The Serine Protease Mechanism in Detail” (p.62~p.65)

5. Substrate selectivity
→利用酵素的立體構造 去適應基質
Elastase:
切立體阻礙小的基質
Trypsin:
切帶正電、長側鏈的基質
Chymotrypsin:
切疏水性的芳香族

6. 呈現黃色!
故可藉顏色變化來評估酵素的活性!
p -Nitrophenylacetate

7. Transition-State Stabilization in the Serine Proteases
• The chymotrypsin mechanism involves
two tetrahedral oxyanion intermediates
• These intermediates are stabilized by a
pair of amide groups that is termed the
“oxyanion hole”

8. 例二: The aspartic protease:
→general acid-base catalysis,
LBHB
• All involve two Asp residues at the active site
• These two Asp residues work together as
general acid-base catalysts
• Most aspartic proteases have a tertiary
structure consisting of two lobes (N-terminal
and C-terminal) with approximate two-fold
symmetry

9. Aspartic Protease Mechanism
LBHB

10. 例三 Chorismate mutase:
Intramolecular rearrangemen
分子內重組

11. Two possible mechanisms of chorismate rearrangement reaction

12. Use TSA to understand the chair mechanism of chorismate mutase
A structure that
resembles a NAC

13. 習題(課本) Tosyl-L-phenylalanine chloromethyl ketone (TPCK)
specifically inhibits chymotrypsin by covalently labeling His57
Q1. State why this inhibitor is
specific for chymotrypsin.
Ans: TPCK is specific for chymotrypsin
because the phenyl ring of the
phenylalanine residue interacts
effectively with the binding pocket of the chymotrypsin active site.
Tosyl-L-phenylalanine chloromethyl ketone (TPCK)
Q2. Propose a reagent based on the structure of TPCK
that might be an effective inhibitor of trypsin.
Ans: Replacement of the phenylalanine residue of
TPCK with arginine or lysine produces reagents that
are specific for trypsin.

14. serine195