● 11 酵素的命名 E1● 22 酵素的構成 E2● 33 酵素動力學 E3● 44 酵素的抑制 E4● 55 酵素的催化機制 E5● 66 酵素活性的調節 E6● 77 細胞代謝與酵素調控 E7● 88 酵素在生物技術上的應用

酵 素EnzymeEnzyme εν ζυημεν ζυημ

基礎

動力機制

調節

應用

HO

H

棒棒脢的催化機制及酸鹼催化Adapted from Nelson & Cox

(2000) Lehninger Principles of

Biochemistry (3e) p.252

誘導生成過渡狀態

CO=

NH

HCH

NH

+

C- OOH

OH

-δ

+δ

HO

H

CO=

NH

HCH

CO=

NH

HCH

CO=

NH

HCH

Slow Fast Fast Very Fast

Acid-baseCatalysis Acidcatalysis

Basecatalysis

Both

Ada

pted

from

Alb

erts

et a

l (20

02) M

olec

ular

Bio

logy

of t

he C

ell (

4e) p

.167

NH

+

C- OO

HO

H

吸引力

5.1 基本反應機制

三個基本動作 1) Bond Strain2) Acid-base transfer3) Orientation

構形扭曲化學轉移空間方向

Carboxypeptidase ACarboxypeptidase BCarboxypeptidase Y

協同式

順序式 ChymotrypsinTrypsinElastase

non-polarRK

non-specific

YFWRKGA

Ser-蛋白脢內切脢

金屬蛋白脢外切脢

Juang RH (2007) BCbasics

協同式外切脢的催化機制

1

2

3 4

5

O-H

+ HCOO-

(270)Glu

(248)Tyr

O-H

His(196)

His (69)

Glu(72)

+Arg (145)

Carboxypeptidase A

C-terminus

ACTIVESITE

ACTIVESITE

C-端確認區

專一性確認區

活性區口袋

基質胜肽鏈

RNCN C

COO-O-

C

+Zn

Juan

g R

H (2

007)

BC

basi

cs

Carboxypeptidase A

Stryer (1995) Biochemistry (4e) p.220

Arg 145

Tyr 248

Glu 270

Stryer (1995) Biochemistry (4e) p.220

加入基質 - 活性區關閉 - 開始催化

Chymotrypsin 的分子模型

Stry

er(1

995)

Bio

chem

istry

(4e)

p.2

07

活性區的三個重要胺基酸

Branden & Tooze (1999) Introduction to Protein Structure (2e) p.212, 210

Chymotrypsin 要先經裂解後才有活性

Ada

pted

from

Cam

pbel

l (19

99) B

ioch

emis

try (

3d) p

.179

245

R15-I16

Chymotrypsinogen (inactive)

π-Chymotrypsin (active)

S14-R15 T147-N148

Trypsin

α-Chymotrypsinogen (active)

π-Chymotrypsin

I16L13 A149Y146

Disulfide bonds

Chymotrypsin 的活性區

Catalytic triad: Asp102←His57←Ser195 charge relaycharge relay(1)(1) 環境 pH 對酵素活性有極大影響 →→ 活性區的 胺基酸 受影響：

(2)(2) 催化機制：兩個步驟

(3)(3) 穩定過渡狀態：-C-O- 可與 Gly193 與 Ser195 的 -N-H 產生氫鍵而穩定之

(4)(4) 專一性結合區：活性區附近有 non-polar pocket 辨識基質

▼ Acylation: 切開後 N-peptide 共價結合在酵素上 (Ser195)▼ Deacylation: 加水分解後釋出 N-peptide (slow step)

Nitrophenyl acetate (作用很慢的基質類似物)

His 57 (pKa = 6): 當環境 pH > 6, imidazole 失去 H+ (charged)Ile 16 (new N-terminal): 當環境 pH > 9, NH3+ 失去 H+ (不帶電)Ser 195: DIFP 可與 Ser-OH 反應 → 失去活性

Juang RH (2007) BCbasics

Chym

otrypsin

活性區的電子接力

Ser195

His 57

Asp 102

H–O–CH2OC–O-

=

Active Ser

H–N N

C C

C

H

H

CH2

Ser195

His 57

Asp 102

-O–CH2OC–O–H

=

N N–H

C C

C

H

H

CH2

Ada

pted

from

Alb

erts

et a

l (20

02) M

olec

ular

Bio

logy

of t

he C

ell (

4e) p

.158

酸鹼度對 Chymotrypsin 活性影響很大

5 6 7 8 9 10 11

pH

相對活性

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162

環境酸鹼度影響蛋白質的淨電荷

Juan

g R

H (2

007)

BC

basi

cs

+Net Charge of a Protein

Buffer pH

Isoelectric point,pI

-

3456789

10

0+

Histidine 上的 imidazole 基團

H–N N

C C

C

H

H

H+

pH < 6 pH > 7

+H–N N–H

C C

C

H

H

Inactive+ Ser

195

His 57

Asp 102

H–O–CH2OC–O-

=

H–N N–H

C C-H

C

CH2

H

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158

Ada

pted

from

Dre

ssle

r & P

otte

r(20

00) D

isco

verin

g E

nzym

es,p

.163

Chymotrypsin 切出新的 Ile16 N-端

I16L13 Y146

Asp 194

–CH2COO-

Ile 16NH2–

Ile 16+NH3–

5 6 7 8 9 10 11pH

相對活性

pH 9 pH 10pKa

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.165

New NH2-terminus

新的 Ile16 N-端可穩定 Asp194

Asp 102

His 57 Ser 195

Asp 194

Gly 193

Ile 16

+NH3

Catalytic TriadCatalytic Triad

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.206

Nelson & Cox (2004) Lehninger Principles of Biochemistry (4e) p.214

I16

S195

D194

O

(CH3)2CH–O– P–O–CH(CH3)2F

=

Chymotrypsin Ser195 被 DIFP 抑制

Diisopropyl-fluorophosphate (DIFP)

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167

O-…HCH2

Ser 195

O

(CH3)2CH–O– P–O–CH(CH3)2

=

O

CH2

Ser 195

XXX

添加基質可抵抗競爭性酵素抑制劑

Reaction time

酵素活性被抑制程度(%

)

100

50

0

不加基質

添加基質

S

+ DIFP

+ DIFP & substrate

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167

XXX

Asp102

His57

Ser195

Catalytic TriadCatalytic Triad

HH

Chymotrypsin 順序式催化反應 A1

NC

CN

[HOOC]H

O

CC

NC

C[NH2]

CC

O

Check substrate specificity

AbzymeAbzyme 催化性抗体

以抗体分子模擬 peptide bond 的水解機制

碳: sp2 → [sp3]* → sp2過渡狀態

Why ?Why ?

Abzyme 與基質有專一性結合能力，但催化效果較真正的酵素差。

以一 sp3 過渡狀態的類似物免疫動物，所得的抗体有水解胜鍵功能。

sp3

Juang RH (2007) BCbasics

酵素可使用類似的反應基質

O-C N-

H

O-C O-

Peptide bond

Ester bond

OCH3–C–O– –NO2

Nitrophenol acetate

HO– –NO2

OCH3–C–OH

Hartley & Kilby

Chymotrypsin+ H2O

Nitrophenol

Acetate

反應初期無法測得 acetate 的生成Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168

O-C

Time (sec)N

itrop

heno

l

證明 Chymotrypsin 兩段式催化反應

OCH3–C–O– –NO2

Nitrophenol acetate

OC

OCH3–C HO– –NO2

+ H2OO-HC

CH3COOH

反應動力學

反應有兩相

Acylation

Deacylation (slow step)

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169

過渡狀態的額外電荷可被穩定

O-C N-

H

O-C-OH

NH2-

-C-C-N-C-C-N-C-C-N-H H

E + S

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.179

O --C N-

HO H

O --C N-

HO H

穩定過渡狀態的分子立體空間

Asp 102

His 57

Met 192

Gly 193

Asp 194Ser 195

Cys 191

Catalytic Triad

Thr 219

Ser 218Gly 216

Ser 217

Trp 215

Ser 214

Cys 220

專一性辨認區

活性區

Ada

pted

from

Dre

ssle

r & P

otte

r(19

91) D

isco

verin

g E

nzym

es,p

.197

O ON–C–C–N–C–C N–C–C–N–C–C

R H R’

Chymotrypsin 活性區另有專一性辨認區

O-CSer

活 性 區活 性 區

專一性辨認區

專一性辨認區 催化區

Juang RH (2007) BCbasics

專一性的形成

●● Trypsin 及其抑制劑： Lys vs Asp (specific binding)↑胰臟 ↑卵白

為何會有如此的專一性？

■■ Trypsin 一族： chymotrypsin, elastase ...Serine proteaseSerine protease → 催化機制相同 專一性不同

●●改變專一性結合區可改變基質專一性 [Science]

Trypsin (Asp189) ← Chymotrypsin (Ser189)

演演 化化Juang RH (2007) BCbasics

Trypsin 家族的專一性不同

COO-CAsp

COO-CAsp

酵素活性區

Trypsin Chymotrypsin Elastase切 Lys, Arg 切 Trp, Phe, Tyr 切 Ala, Gly

Non-polarpocket

Dee

p an

d ne

gativ

ely

char

ged

pock

et Shallow andnon-polar

pocket

O O–C–N–C–C–N–

CCCCNH3+

O O–C–N–C–C–N–

C

O O–C–N–C–C–N–

CH3

Juan

g R

H (2

007)

BC

basi

cs

TrypsinAsp 189

ChymotrypsinSer 189 Asp

HisSer

可以改變酵素的專一性

Ester (+)Amide (-)

Hedstrom et al (1992) Science 255: p.1250

定點突變

蛋白質間專一性的形成

胰蛋白脢

抑制劑

I. Conformational Match:Van der waals interaction

II. Interaction Forces:(1) Hydrogen bond(2) Hydrophobic interaction(3) Electrostatic interaction(4) Van der waals interaction

+Kd

Stry

er(1

995)

Bio

chem

istry

(4e)

p.2

52

Juan

g R

H (2

007)

BC

basi

cs

5.4.3 酵素的立體專一性

BC

DB CDB C

D

這兩個三角形並不相同

A

碳原子的四面體構造有很強的立體限制性是蛋白質構形的根本

Juang RH (2007) BCbasics

sp3

酵素表面

以下說明幾個重要催化實例，說明酵素的作用機制與活性區之組成。

這幾張投影片在網站特附有 PowerPoint 檔案 Enz(5A)，可看到動畫。

Substrate binding site

+

Lysozyme 活性區

E35

C

O

O-H

D52

C

O

O-O

:O

O

D

E

E35

C

O

O-

D52

C

O

O-

H-O

OE

DO

H-O-H:

E35

C

O

OH

D52

C

O

O-

O D

OH

Electrostatic catalysis

Non-polarenvironment Polar

environment

First product

N-acetylglucosamine(NAG)

N-acetylmuramic acid(NAM)

NAG-NAM-NAG-NAM-NAG-NAM-

A B C D E F

Stryer (2002) Biochemistry (5e) p.199, Fig 8-7

兩個酸基在 pH 5 下有不同形式

Chair → Half-chair

H-bonds

O

O

sp3

sp2

sp2

Catalytic toolkit for active sites

Gutteridge A, Thornton JM (2005) Understanding nature’s catalytic toolkit. TIBS 30: 622

Combinations of different residues form ‘catalytic units’that are found repeatedly in different unrelated enzymes

HistidineHistidineHistidine

三連 Arg-Arg-Arg 正電集團 Asp-Asp 酸鹼配對 (Thr 定位) Asp-Arg 離子配對 (影響 pKa) Arg…Asp-COO- (親核攻擊)

Asp-Lys-Thr triad (reactive -O-) Asp-Lys 配對 (調節 Tyr pKa) KEK (EK 控制 K112 pKa) 簡單的酸鹼對 (質子供需)

Arg, carboxlyate

Lysine

Histidine

Asp-His-Ser triad (reactive -O-) Asp-His 配對 (攻擊基質) His-His dyad (H83 攻擊磷酸)

Amazing histidine被採用數目

被採用頻率

Gutteridge A, Thornton JM (2005) TIBS 30: 622 WIKIPEDIA

H57

S195

D102

Petsko GA, Ringe D (2004) Protein Structure and Function F4-35

Ribonuclease

:N+ NH

H12

HN N+-H

H119

O

OPO2-OR3’

R5’ Base

OH

OR3’

PO2-HN N+-H

H119N+ NH

H12

H

O

O

R5’ Base

O

HN N:

H119N+ NH

H12

HPO2-

O

O

R5’ Base

O

H-O-H

:N+ NH

H12

HN N+-H

H119

O

OPO2-OH

R5’ Base

OH

First productHistidine proton shuffle

HOR3’

質子供應者

質子強奪者

Multifunctional enzymes

(1) One active site, two reactions(2) Two active sites, two reactions(3) Trifunctional enzyme with tunnel

Carbamoyl phosphate synthetase →

Tryptophan synthase↓

Petsko GA, Ringe D (2004) Protein Structure and Function F2-44, 45

演化真是無限可能

Enzymes also has non-catalytic functionsZheng, L et al (2003) S phase activation of the histone H2B promoter by OCA-S, a coactivatorcomplex that contains GAPDH as a key component. Cell 114: 255~266

GAPDH has several functions:(0) Glycolysis enzyme(1) Transcription cofactor(2) Initiates apoptosis(3) ER to Golgi transportation

Other examples:Phosphoglucose isomerase

(Glycolysis & Cytokine)

LON (Mitochondrial protease

& Chaperone protein)

WIKIPEDIA

Glyceraldehyde-3-phosphate dehydrogenase