Download Presentasi 1

Basic enzyme
Aulanni’am
Biochemistry Laboratory
Brawijaya University
Aulani " Biokimia Enzim " Presentasi 1
1
What are enzymes ?




Enzymes are proteins
They have at least one active site
Active site is lined with residues and sometimes contains a
co-factor
Active site residues have several important properties:
 Charge [partial, dipoles, helix dipole]
 pKa
 Hydrophobicity
 Flexibility

Reactivity (Cysteines)
Aulani " Biokimia Enzim " Presentasi 1
2
What are chemical reactions?






In a chemical reactions a compound “A” is changed into a
compound “B”.
In context of biochemistry, chemical reactions are
“organic chemistry reactions”.
In organic chemistry reactions bonds are broken and/or
formed (generalization)
Bonds are “paired electrons” between two nuclei (C-C, C=C,
C-O,C=O, C-H, O-H, N-H etc.)
Thus reactions involve “rearranging” electrons
In context of biochemistry, a frequent player in chemical
reactions is H2O (hydronium H3O+ and hydroxide OH-)
Aulani " Biokimia Enzim " Presentasi 1
3
Enzyme catalysis


Enzyme catalysis is characterized by two
features
Substrate specificity
Rate acceleration
Aulani " Biokimia Enzim " Presentasi 1
4
Enzyme substrate specificity
Unlike “chemical catalysts” enzyme only catalyze reactions
for a “relatively” narrow substrate spectrum.
For example: substrate spectrum of restriction enzymes,
and protein kinases.
Two main theories for substrate specificity
 Lock-and-Key hypothesis (Fisher, 1894)
 Induced-fit hypothesis (Koshland, 1958)
Aulani " Biokimia Enzim " Presentasi 1
5
Substrate
Transition state
X
Product
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
Aulani " Biokimia Enzim " Presentasi 1
6
The Nature of Enzyme Catalysis
● Enzyme provides a catalytic surface
● This surface stabilizes transition
state
● Transformed transition state to product
B
A
A
B
Aulani " Biokimia Enzim " Presentasi 1
Catalytic surface
7
Lock-and-Key vs. Induced-Fit


Lock-and-Key does not always explain substrate
spectrum (e.g. analogs smaller than substrate don’t bind
while analogs larger than substrate do bind)
Induced-fit implies the concepts:
 conformational change
 catalytically competent conformation (low catalytic
form and high catalytic form)
Aulani " Biokimia Enzim " Presentasi 1
8
Catalyzed vs. un-catalyzed reactions
Free Energy (delta G)
S‡
S‡c
ES‡
S
ES
EP
P
Reaction Coordinate
Aulani " Biokimia Enzim " Presentasi 1
9
Specific
Induced to transition state
+
N
H
N
H
=
O
C H
+d
+
Acid
catalysis
C
H
N
H
=
N
H
C
O H
H
H
-d
O
H
O
C H
=
N
H
Base
catalysis
Fast
C
H
O
H
H
Slow
O
C H
O
H
-
O
O
C H
=
Acid-base
Catalysis
Both
N
C
H O H
H
H
O
-
O
C
C
Fast
Very Fast
Aulani " Biokimia Enzim " Presentasi 1
10
Rate Acceleration



Catalyzes of a reaction results in
rate enhancement not alteration of
the equilibrium
Catalysis involves reduction of
activation energy
This can be most readily done by
lowering the Free Energy of the
transition state
Additionally the Free Energy of the
ground state can be raised (not a
general strategy)
Free Energy (delta G)

S‡
ES‡
S
ES
EP
P
Reaction Coordinate
Aulani " Biokimia Enzim " Presentasi 1
11
Transition state Stabilization by Enzyme
S‡
Free Energy (delta G)
How does an Enzyme reduce the
Activation Energy ??
 Enzyme stabilizes the transition
state, i.e. makes the “strained”
conformation more bearable.
Note:
 An enzyme can only reduce the
activation energy if it binds better
to the transition state than to the
substrate
[otherwise, the DDG between ES
and ES‡ is the same as between S
and S‡]
Aulani " Biokimia Enzim " Presentasi 1
ES‡
S
ES
EP
P
Reaction Coordinate
12
Transition state Stabilization by Enzyme


Compounds that closely mimic the
transition state bind much better to
an enzyme than the original
substrate.
Transition state analogs are potent
inhibitors (pico molar affinities)
Free Energy (delta G)
Implications of preferential
stabilization of the transition state.
S‡
ES‡
S
ES
EP
P
Reaction Coordinate
Applications:
• Inhibitor/drug development based on transition state model
• Development of catalytic antibodies [rate acceleration up to 105]
Aulani " Biokimia Enzim " Presentasi 1
13
Enzyme Stabilizes Transition State
Energy change
EST
S
ES
EP
P
Energy decreases (under catalysis)
Energy required (no catalysis)
T = Transition state
ST
Reaction direction
What’s the difference?
Aulani " Biokimia Enzim " Presentasi 1
14
Active Site Is a Deep Buried Pocket
Why energy required to reach transition state is lower in
the active site?
It is a magic pocket
(1) Stabilizes transition
+
CoE (1)
(4)
-
(2) Expels water
(2)
(3) Reactive groups
(4) Coenzyme helps
(3)
Aulani " Biokimia Enzim " Presentasi 1
15
Enzyme Active Site Is Deeper than Ab Binding
Ag binding site on Ab binds to Ag
complementally, no further reaction
occurs.
Instead, active site on enzyme
also recognizes substrate, but
actually complementally fits the
transition state and stabilized it.
X
Aulani " Biokimia Enzim " Presentasi 1
16
Enzyme mediated catalysis


Strategies for transition state stabilization
and/or ground state destabilization:
 Proximity
 Strain or distortion
 Orbital steering
However, additionally the enzyme can be an
“active” participant in reaction
 Acid/base catalysis
 Nucleophilic/electrophilic catalysis
 Covalent catalysis
Aulani " Biokimia Enzim " Presentasi 1
17
Rate Acceleration: Proximity




For un-catalyzed reactions involving two substrates the
rate can be increased by increasing the number of
collisions (higher temperature)
Enzymes capture each substrate (sometimes in a ordered
manner) and appropriately orient them with respect to
each other, thus obviating the need for higher
temperature
The capture of substrates by the enzyme has an Entropic
cost; this cost must be compensated by favourable
interactions between enzyme and substrates
The effect of confining the substrates in the active site
of the enzyme is similar to raising the concentration of
the substrates. Hence, the proximity effect is also
referred to as increasing the effective concentration
Aulani " Biokimia Enzim " Presentasi 1
18
Active Site Avoids the Influence of Water
+
Preventing the influence of water sustains the formation of stable ionic bonds
Aulani " Biokimia Enzim " Presentasi 1
19
Essential of Enzyme Kinetics
Steady State Theory
E
+
S
E
S
E +P
In steady state, the production and consumption of the transition state proceed at the
same rate. So the concentration of transition state keeps a constant.
Aulani " Biokimia Enzim " Presentasi 1
20
Constant ES Concentration at Steady State
S
P
Concentration
E
ES
Reaction Time
Aulani " Biokimia Enzim " Presentasi 1
21
The “Active” Enzyme
Examine the hydrolysis of an ester:
O
R'
R
O
very slow
+
+
H2O
O
Weak electrophile
R
R'
OH
Poor nucleophile
O
O
Expected
transition state
HO
R'
R
O
H
R'
R
O
O
H
O
d
d
H
H
Aulani " Biokimia Enzim " Presentasi 1
22
The “Active” Enzyme
Base catalyzed hydrolysis of an ester:
O
O
O
R'
R
O
+
R
-
O
+
R'
OH
R
HO
O-
OH
Catalysis is accelerated by altering the poor nucleophile H2O into a
strong nucleophile OH-
Aulani " Biokimia Enzim " Presentasi 1
23
R'
The “Active” Enzyme
Acid catalyzed hydrolysis of an ester:
+
OH
O
R'
R
+
H3O+
OH
R'
R
O
C+
O
R
R'
O
d
OH
C
R
d
O
R'
+
O
R
HO
R'
OH
O
H
H
Catalysis is accelerated by altering the weak electrophile C into a strong
nucleophile C+
Aulani " Biokimia Enzim " Presentasi 1
24
The “Active” Enzyme



In standard organic chemistry for ester hydrolysis
one has to choose between base or acid catalysis
In enzyme catalysis the reaction is “carried out on a
solid support”
As a consequence one can incorporate both acid and
base catalysis:
Aulani " Biokimia Enzim " Presentasi 1
25
The “Active” Enzyme
Enzyme catalyzed
hydrolysis of an ester:
B+
H
Active site incorporates
both:
O
R'
R
• a base [-B:]
O
O
H
H
B:
• an acid [-B+-H]
B:
B+
B:
H
d
OH
O
C
R'
R
R
O
H
B:
H
B+
H
d
R'
C
O
-
O
O
HO
R
O
O
H
Aulani " Biokimia Enzim " Presentasi 1
B+
H
H
26
R'
Catalysis of Phosphorylation



Phosphorylation a very frequent reaction (e.g. signal
transduction)
Phosphoryl donating group is generally a nucleotide,
e.g. ATP, GTP
Transfer of phosphoryl group to:
 Water : hydrolysis [ATPase, GTPase]
 Anything else: phosphorylation [Kinase]
Aulani " Biokimia Enzim " Presentasi 1
27
Mechanisms of Enzyme Catalyzed
Phosphorylation


Several mechanism are observed in Nature
 Reactions with covalent enzyme intermediates
 Direct inline transfer
 Perhaps metal assisted mechanisms
Present two examples:
 Aminoglycoside kinases (Cousin of Protein kinases)
 G-proteins
Aulani " Biokimia Enzim " Presentasi 1
28
An Example for Enzyme Kinetics (Invertase)
1) Use predefined amount of Enzyme
→E
→ S (x
2) Add substrate in various concentrations
3) Measure Product in fixed Time (P/t) → vo (y
4) (x, y) plot get hyperbolic curve, estimate
→ Vmax
5) When y = 1/2 Vmax calculate x ([S])
→ Km
Vmax
1
vo
-1
Km
vo
1/2
1
Vmax
Double reciprocal
1/S
Aulani " Biokimia Enzim " Presentasi 1
Km Direct plot S
29
A Real Example for Enzyme Kinetics
Data
Substrate Product
Velocity
Double reciprocal
[S] Absorbance v (mmole/min)
0.25
0.21 →
0.42
0.50
0.36 →
0.72
1.0
0.40 →
0.80
2.0
0.46 →
0.92
no
1
2
3
4
1/S
4
2
1
0.5
1/v
2.08
1.56
1.35
1.16
Double reciprocal
Direct plot
(1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole
(2) Reaction time was 10 min
1.0
v
0.5
0
0
1
2
[S]
2.0
1.0
1/v
1.0
-3.8
0
-4
Aulani " Biokimia Enzim " Presentasi 1
-2
0
2
1/[S]
4
30
Enzyme Inhibition (Mechanism)
Equation and Description
Cartoon Guide
I
Competitive
I
Non-competitive
Substrate
E
S
S
E
I
Compete for
Inhibitor active site
S
I
I
Uncompetitive
S
E
I
I
Different site
E + S←
→ ES → E + P
+
I
↓↑
EI
E + S←
→ ES → E + P
+
+
I
I
↓↑
↓↑
EI + S →EIS
[I] binds to free [E] only,
and competes with [S];
increasing [S] overcomes
Inhibition by [I].
[I] binds to free [E] or [ES]
complex; Increasing [S] can
not overcome [I] inhibition.
Aulani " Biokimia Enzim " Presentasi 1
S
I
E + S←
→ ES → E + P
+
I
↓↑
EIS
[I] binds to [ES] complex
only, increasing [S] favors
the inhibition by [I].
31
Enzyme Inhibition (Plots)
I
Competitive
I
Non-competitive
Direct Plots
Vmax
vo
vo
I
Double Reciprocal
Km Km’
I
[S], mM
Km = Km’
I
Uncompetitive
Vmax
Vmax
Vmax’
Vmax’
[S], mM
I
Km’ Km
[S], mM
Vmax unchanged
Km increased
Vmax decreased
Km unchanged
Both Vmax & Km decreased
1/vo
1/vo
1/vo
Intersect
at Y axis
1/Km
I
I
I
Two parallel
lines
1/ Vmax
1/[S]
Intersect
at X axis
1/Km
1/ Vmax
1/[S]
Aulani " Biokimia Enzim " Presentasi 1
1/ Vmax
1/Km
1/[S]
32
H
=
O
C–O -
H–N
C
N
H–O–CH2
C C
H
CH2
Asp 102
Ser
195
His 57
Active Ser
H
=
O
C–O–H
Asp 102
N
C
N–H
- O–CH
C C
H
CH2
2
Ser
195
His 57
Aulani " Biokimia Enzim " Presentasi 1
33
pH Influences Chymotrypsin Activity
Relative Activity
5
6
7
8
9
10
11
pH
Aulani " Biokimia Enzim " Presentasi 1
34
Buffer pH
pH Influences Net Charge of Protein
10
9
8
7
Isoelectric point,
pI
+
+
6
5
4
3
0
Net Charge of a Protein
Aulani " Biokimia Enzim " Presentasi 1
35
Imidazole on Histidine Is Affected by pH
pH 6
H
H–N
C
C
+
N–H
H+
H–N
C
C
H
=
Asp 102
H–N
C
C
N
C
H
H
O
C–O -
pH 7
H
Inactive
+
N–H H–O–CH2
C C-H
CH2
Ser
195
His 57
Aulani " Biokimia Enzim " Presentasi 1
36
Chymotrypsin Produces New Ile16 N-Terminal
New NH2-terminus
Relative activity
L13
pH 5
6
7
8
I16
Y146
9 10 11
NH2– Ile 16
Asp 194
pH 9
–CH2COO+ NH
3–
pKa
pH 10
Ile 16
Aulani " Biokimia Enzim " Presentasi 1
37
New Ile16 N-Terminal Stabilizes Asp194
Catalytic Triad
His 57
Asp 102
Ser 195
Gly 193
Asp 194
+NH
3
Ile 16
Aulani " Biokimia Enzim " Presentasi 1
38
Chymotrypsin Ser195 Inhibited by DIFP
Diisopropyl-fluorophosphate (DIFP)
X
=
O
(CH3)2CH–O– P –O–CH(CH3)2
F
=
O
(CH3)2CH–O– P –O–CH(CH3)2
O-…H
O
CH2
CH2
Ser 195
Ser 195
Aulani " Biokimia Enzim " Presentasi 1
39
Addition of Substrate Blocks DIFP Inhibition
100
No substrate
Percent Inhibition of activity (%)
+ DIFP
X
50
+ DIFP & substrate
Add substrate
S
0
Reaction time
Aulani " Biokimia Enzim " Presentasi 1
40
Chymotrypsin Also Catalyzes Acetate
Hartley & Kilby
O
-C NH
Nitrophenol acetate
O
CH3–C–O–
+ H 2O
–NO2
Chymotrypsin
Peptide bond
O
-C OEster bond
HO–
–NO2
Acetate
O
CH3–C–OH
Nitrophenol
No acetate was detected at early stage
Aulani " Biokimia Enzim " Presentasi 1
41
Two-Stage Catalysis of Chymotrypsin
O Nitrophenol acetate
CH3–C–O–
–NO2
Acylation
O
CH3–C
–NO2
O
C
O-H
C
+ H2O
Aulani " Biokimia Enzim " Presentasi 1
Nitrophenol
CH3COOH
Deacylation (slow step)
Kinetics of reaction
OC
HO–
Two-phase
reaction
Time (sec)
42
Extra Negative Charge Was Neutralized
-C-C-N-C-C-N-C-C-NH
H
O-C NHO H
O-C NHO H
O
-C NH
E+S
O
-C-OH
NH2-
Aulani " Biokimia Enzim " Presentasi 1
43
Active Site Stabilizes Transition State
Gly 193
Catalytic Triad
Ser 195
Asp 194
Met 192
His 57
Active Site
Cys 191
Asp 102
Thr 219
Cys 220
Specificity Site
Ser 214
Trp 215
Gly 216
Ser 218
Ser 217
Aulani " Biokimia Enzim " Presentasi 1
44
Regulation of Enzyme Activity
Inhibitor
Proteolysis
or
o
I
x
I
I
S
proteolysis
x
I
o
S
inhibitor
Feedback regulation
R
o
Phosophorylation
P
x
x
o
S
R
S
regulator
effector
(+)
P
phosphorylation
Signal transduction
x
(-)
A or
Regulatory
subunit
o
S
+
Aulani " Biokimia Enzim " Presentasi 1
A
cAMP or
calmodulin
45
Classification of Proteases
Family
Metal
Protease
Serine
Protease
Cysteine
Protease
Aspartyl
Protease
Example
Carboxypeptidase A
Chymotrypsin
Trypsin
Mechanism
Zn
Specificity Inhibitor
2+
E72 H69
H196
S195-OH57
D102
C25-S-
Nonpolar
EDTA
EGTA
Aromatic
Basic
DFP
TLCK
TPCK
PCMB
Leupeptin
Pepstatin
Papain
H195
Nonspecific
Pepsin
Renin
D215
H2O
D32
Nonspecific
Aulani " Biokimia Enzim " Presentasi 1
46
Ser 195
Chymotrypsin
Trypsin
Elastase
Thrombin
Plasmin
Acetylcholinesterase
– Gly – Asp – Ser – Gly – Gly – Pro – Leu –
– Gly – Asp – Ser – Gly – Gly – Pro – Val –
– Gly – Asp – Ser – Gly – Gly – Pro – Leu –
– Gly – Asp – Ser – Gly – Gly – Pro – Phe –
– Gly – Asp – Ser – Gly – Gly – Pro – Leu –
– Gly – Glu – Ser – Ala – Gly – Gly – Ala –
His 57
Chymotrypsin
Trypsin
Elastase
Thrombin
Plasmin
Acetylcholinesterase
– Val – Thr – Ala – Ala – His – Cys – Gly –
– Val – Ser – Ala – Gly – His – Cys – Tyr –
– Leu – Thr – Ala – Ala – His – Cys – Ile –
– Leu – Thr – Ala – Ala – His – Cys – Leu –
– Leu – Thr – Ala – Ala – His – Cys – Leu –
– – – – – – – – – – – – – His – – – – – – – –
Asp 102
Serine Protease and AchE
Chymotrypsin
Trypsin
Elastase
Thrombin
Plasmin
Acetylcholinesterase
– Thr – Ile – Asn – Asn – Asp – Ile – Thr –
– Tyr – Leu – Asn – Asn – Asp – Ile – Met –
– Ser – Lys – Gly – Asn – Asp – Ile – Ala –
– Asn – Leu – Asp – Arg – Asp – Ile – Ala –
– Phe – Thr – Arg – Lys – Asp – Ile – Ala –
– – – – – – – – – – – – – – Asp – – – – – – –
Aulani " Biokimia Enzim " Presentasi 1
47
Sigmoidal Curve Effect
vo
Sigmoidal curve
Noncooperative
(Hyperbolic)
ATP
Positive effector (ATP)
brings sigmoidal curve
back to hyperbolic
CTP
Cooperative
(Sigmoidal)
Negative effector
(CTP)
keeps
vo
Exaggeration of
sigmoidal curve
yields a drastic
zigzag line that
shows the On/Off
point clearly
Consequently,
Allosteric enzyme
can sense the
concentration of
the environment and
adjust its activity
Off
[Substrate]
On
Aulani " Biokimia Enzim " Presentasi 1
48
Mechanism and Example of Allosteric Effect
Kinetics
R = Relax
(active)
Models
Cooperation
Allosteric site
Homotropic
R
vo
(+)
S
S
R
S
[S]
R
Concerted
Allosteric site
A
(+)
vo
S
(+)
Heterotropic
T
(+)
S
Sequential
R
X
[S]
T
T = Tense
(inactive)
(+)
I
vo
(-)
X
(-)
T
[S]
Aulani " Biokimia Enzim " Presentasi 1
T
X
Heterotropic
(-)
Concerted
49
Activity Regulation of Glycogen Phosphorylase
Covalent modification
P
A
P
GP phosphatase 1
AAP
T
spontaneously
AMP
Non-covalent
ATP
Glc-6-P
Glucose
Caffeine
A
Glucose
Caffeine
A
P
P
P
A
P
R
P
R
A
P
T
GP kinase
A
P
A
P
A
P
A
Aulani " Biokimia Enzim " Presentasi 1
50
Aulani " Biokimia Enzim " Presentasi 1
51