MOLECULAR MECHANISMS

OF ENZYME ACTION

(WEEK 7)
 MOLECULAR MECHANISMS OF
ENZYME ACTION
1. Understanding molecular mechanisms of enzyme action
2. Molecular mechanisms of chymotripsin-enzyme action
3. Molecular mechanisms of DNA ligase-enzyme action
4. Molecular mechanisms of ATPase-enzyme action
5. Molecular mechanisms of aspartate transkarbamoylase-enzyme action
6. Molecular mechanisms of choline esterase-enzyme action
7. Molecular mechanisms of papain (sistein protease)-enzyme action
8. Molecular mechanisms of para-hydroxybenzoate hydroxylase (PHBH)-enzyme
action
9. Molecular mechanisms of HIV integrase-enzyme action
10.Molecular mechanisms of alcohol dehidrogenase-enzyme action
 KATALISIS OLEH ENZIM
CHYMOTRYPSIN
Chymotripsin mengkatalisis hidrolisis ikatan
peptida.
Hidrolisis amida dengan katalis chymotrypsin
dicapai dengan sebuah ion karboksilat yang
berperan sebagai basa.
Ion karboksilat terdapat didalam cavity enzim
dekat dengan sisi aktifnya sehingga konsentrasi
efektifnya lebih tinggi daripada jika basa
seperti Na-asetat ditambahkan dalam larutan.
 Acid-Base Catalysis

Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.167

Specific Induced to transition state

Acid-base
Catalysis + Acid +
catalysis
N N
O H H
O
=

C O O

=
+ H C

=
=

N C C H Both C
H H H N C H
N C H O H N C
H O H H O H
- H H H H
O H H
H H O - O O - O
Base C C
catalysis
Slow Fast Fast Very Fast
 Chymotrypsin Is Activated by Proteolysis
Chymotrypsinogen (inactive)

245

Trypsin
-Chymotrypsin (active)
R15-I16

Adapted from Campbell (1999) Biochemistry (3d) p.179

-Chymotrypsin

S14-R15 T147-N148

L13 I16 Y146 A149

Disulfide bonds
-Chymotrypsinogen (active)
Charge Relay in Active Site
H
O
C

=
- HN N HOCH2 Ser
CO
195
C C

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158
Asp 102 H
CH2

His 57

H Active Ser
O
C
=

COH N NH - Ser
OCH2
195
C C
Asp 102 H
CH2

His 57
 Stickase
Substrate

Transition state Product

X
If enzyme just binds substrate
then there will be no further reaction

Enzyme not only recognizes substrate,

but also induces the formation of transition state
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
 The Nature of Enzyme Catalysis

Enzyme provides a catalytic surface

This surface stabilizes transition state
Transformed transition state to product

B
A

A B Catalytic surface
Juang RH (2004) BCbasics
 Enzyme Stabilizes Transition State
Energy change
ST
Energy required (no catalysis)

Energy decreases (under catalysis)

EST
S

ES
EP P

Reaction direction
T = Transition state Whats the difference?
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
 Active Site Is a Deep Buried Pocket

Why energy required to reach transition state

is lower in the active site?
It is a magic pocke

+ (1) Stabilizes transition

(2) Expels water
CoE (1) (2)
(3) Reactive groups
(4) - (4) Coenzyme helps
(3)
Juang RH (2004) BCbasics
Enzyme Active Site Is Deeper than Ab Binding

Instead, active site on enzyme

also recognizes substrate, but
Ag binding site on Ab binds to Ag
actually complementally fits the
complementally, no further reaction
transition state and stabilized it.
occurs.

Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
 Active Site Avoids the Influence of Water

+
-

Preventing the influence of water sustains the formation of stable ionic bonds
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115
Chymotrypsin Has A Site for Specificity

O O
NCCNCC NCCNCC
R H R
O-
C
Ser
Specificity
Site Catalytic Site

Active Site

Juang RH (2004) BCbasics

 Specificity of Ser-Protease Family
Trypsin Chymotrypsin Elastase
cut at Lys, Arg cut at Trp, Phe, Tyr cut at Ala, Gly

O O
O O O O
CNCCN
CNCCN CNCCN
C
C
C CH3
C
Deep and negatively
charged pocket

C Shallow and
NH3 non-polar
+ pocket
COO-

Juang RH (2004) BCbasics

Non-polar
C pocket
Asp

Active Site
 Stereo Specificity

A
sp3
D
B D C
Enzyme surface C B B
The tetrahedral structure C D
of carbon orbital has rigid
steric strain which makes
the basic building unit of These two triangles are
protein conformation not identical
Juang RH (2004) BCbasics
 Active Site Stabilizes Transition State

Gly 193
Catalytic Triad
Ser 195 Asp 194

Met 192

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.197

His 57
Active Site
Cys 191
Asp 102

Thr 219
Cys 220

Ser 214
Specificity Site
Trp 215

Gly 216 Ser 218

Ser 217
XIII MOLECULAR MECHANISMS OF ENZYME (CHYMOTRYPSIN) REACTION
Binding of substrate
Note charge-relay system
occurs in anhydrous active
site (protective environment)

Nucleophilic attack by Ser O-

Note tetrahedral C and
formation of H bonds
between substrate and
oxyanion hole. Gly
193 and Ser195
contribute. This is
stabilization of
intermediate.
Product with new N-
terminus is released.

New acyl intermediate

(C no longer
tetrahedral)
Water enters to stabilize partial negative charge on C
A second tetrahedral
intermediate forms
New C-terminal
product is formed
Product is released
and enzyme is ready
for next round of
catalysis.
ANIMATED MODEL OF ENZYME (CHYMOTRYPSIN) REACTION WITH TWO
TRANSITION STATES
Chymotrypsin Catalytic Mechanism A1

Catalytic Triad
His57

Asp102 Ser195
H H
O

[HOOC] C N C C [NH2]
N C H C N C
C
O

Check substrate specificity

Chymotrypsin Catalytic Mechanism A2

His57

Asp102 Ser195
H H
O

C C [NH2]
C N
[HOOC] C N C
N C H C
O

First Transition State

 Chymotrypsin Catalytic Mechanism A3

H O
H
C C
C [NH2 ]
C N C N
[HOOC] C
N C H O

Acyl-Enzyme Intermediate
 Chymotrypsin Catalytic Mechanism D1

H O
O
C C
H
C [NH2 ]
C N C
O

C N-H
[HOOC]
N C H

Acyl-Enzyme Water Intermediate

Chymotrypsin Catalytic Mechanism D2

H
O
C C
O C [NH2 ]
H C N C
O

Second Transition State

Chymotrypsin Catalytic Mechanism D3

H
H O

O C C [NH2]
H C N C
C
O

Deacylation
 Concerted Mechanism of Catalysis
Carboxypeptidase A
(248)
Active (270)
site Tyr
Glu 3 4 ACTIVE
pocket O - Site for
COO - H SITE specificity
H
+
H O-
C N R 5
N C C
2
Substrate O-

Juang RH (2004) BCbasics

peptide 1 + COO - +
Arg (145)
chain His Zn Glu C-terminus
(196) (72)
His (69) Check for
C-terminal
 AMINOACYL-tRNA SYNTHETASE

Enzyme-tyrosine-
ATP complex

Tyrosyl-
AMP

Tyrosyl-tRNA synthetase
(a)

Enzyme-tyrosyl-
AMP complex

Transition state
(b)
 pH Influences Chymotrypsin Activity

Relative Activity

5 6 7 8 9 10 11
pH
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162
 Buffer pH
pH Influences Net Charge of Protein

10
9
8
7
Isoelectric point, 6
pI
5
4

Juang RH (2004) BCbasics

3

+ + 0 -
Net Charge of a Protein
 Imidazole on Histidine Is Affected by pH

Adapted from Dressler & Potter (2000) Discovering Enzymes, p.163

pH 6 H H pH 7
C + C
HN NH HN N
H +

C C C C
H H

H
O Inactive
C +
=

HN NH HOCH2 Ser
CO -
195
C C-H
Asp 102
CH2

His 57

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158
Chymotrypsin Produces New Ile16 N-Terminal
New NH2-terminus
Relative activity

L13 I16 Y146

pH 5 6 7 8 9 10 11

NH2 Ile 16

Asp 194
pKa
pH 9 pH 10
CH2COO -

+
NH3 Ile 16
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.165
 New Ile16 N-Terminal Stabilizes Asp194
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.206

Catalytic Triad
Gly 193
His 57 Ser 195

Asp 102 Asp 194

+
NH3

Ile 16

Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.112

 Chymotrypsin Ser195 Inhibited by DIFP

Diisopropyl-fluorophosphate (DIFP)

O
=
X
(CH3)2CHO P OCH(CH3)2
F
O

=
(CH3)2CHO P OCH(CH3)2
O-H O
CH2 CH2

Ser 195 Ser 195

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

 Addition of Substrate Blocks DIFP Inhibition

100
No substrate
+ DIFP
Percent Inhibition of activity (%)

X
50

+ DIFP & substrate Add substrate

S
0
Reaction time
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167
 Chymotrypsin Also Catalyzes Acetate
Hartley & Kilby
Nitrophenol acetate
O
O CH3CO NO2
-C N-
H + H 2O Chymotrypsin
Peptide bond

O HO NO2
Acetate
-C O- O Nitrophenol
CH3COH
Ester bond
No acetate was detected at early stage
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168
 Two-Stage Catalysis of Chymotrypsin
O Nitrophenol acetate Acylation
O
CH3CO NO2
CH3C HO NO2

O- O

Kinetics of reaction
C C

Deacylation (slow step)

CH3COOH
+ H2 O

Nitrophenol
O-H Two-phase
C reaction

Time (sec)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169
Extra Negative Charge Was Neutralized
-C-C-N-C-C-N-C-C-N-
H H

O- O-
-C N- -C N-
HO H HO H

O
-C N-
H

E+S O
-C-OH

NH2-
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.179
 Active Site Stabilizes Transition State

Gly 193
Catalytic Triad
Ser 195 Asp 194

Met 192

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.197

His 57
Active Site
Cys 191
Asp 102

Thr 219
Cys 220

Ser 214
Specificity Site
Trp 215

Gly 216 Ser 218

Ser 217