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Basic enzyme. Aulanni’am Biochemistry Laboratory Brawijaya University. 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: - PowerPoint PPT Presentation
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Aulani " Biokimia Enzim " Presentasi 1 1
Basic enzyme
Aulanni’am
Biochemistry Laboratory
Brawijaya University
Aulani " Biokimia Enzim " Presentasi 1 2
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 3
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 4
Enzyme catalysis
Enzyme catalysis is characterized by two features
Substrate specificity Rate acceleration
Aulani " Biokimia Enzim " Presentasi 1 5
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 6
Substrate
If enzyme just binds substrate then there will be no further reaction
Transition state Product
Enzyme not only recognizes substrate, but also induces the formation of transition state
X
Aulani " Biokimia Enzim " Presentasi 1 7
The Nature of Enzyme Catalysis
●● Enzyme provides a catalytic surface
●● This surface stabilizes transition state
●● Transformed transition state to product
B
BA Catalytic surface
A
Aulani " Biokimia Enzim " Presentasi 1 8
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 9
Catalyzed vs. un-catalyzed reactions
Reaction CoordinateReaction Coordinate
Fre
e E
nerg
y (d
elta
G)
S
P
S‡
S‡c
ES‡
ES
EP
Aulani " Biokimia Enzim " Presentasi 1 10
HO
H
Induced to transition state
CO=
NH
HCH
NH
+
C- OOH
OH
-
+
HO
H
CO=
NH
HCH
CO=
NH
HCH
CO=
NH
HCH
Slow Fast Fast Very Fast
Acid-baseCatalysis Acid
catalysis
Basecatalysis
Both
NH
+
C- OO
HO
H
Specific
Aulani " Biokimia Enzim " Presentasi 1 11
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)
S‡
ES‡
ES
EP
Reaction Coordinate
Fre
e E
nerg
y (d
elta
G)
S
P
Aulani " Biokimia Enzim " Presentasi 1 12
Transition state Stabilization by Enzyme
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‡]
S‡
ES‡
ES
EP
Reaction CoordinateF
ree
Ene
rgy
(del
ta G
)
S
P
Aulani " Biokimia Enzim " Presentasi 1 13
Transition state Stabilization by Enzyme
Implications of preferential stabilization of the transition state.
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)
S‡
ES‡
ES
EP
Reaction Coordinate
Fre
e E
nerg
y (d
elta
G)
S
P
Applications:
• Inhibitor/drug development based on transition state model
• Development of catalytic antibodies [rate acceleration up to 105]
Aulani " Biokimia Enzim " Presentasi 1 14
Enzyme Stabilizes Transition State
S
P
ES
EST
EP
ST
Reaction direction
Energy change
Energ
y re
quire
d (n
o
cata
lysis)
Energ
y d
ecre
ase
s (under
cata
lysis)
What’s the difference?T = Transition state
Aulani " Biokimia Enzim " Presentasi 1 15
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
(2) Expels water
(3) Reactive groups
(4) Coenzyme helps
(2)
(3)(4)
(1)CoE
+
-
Aulani " Biokimia Enzim " Presentasi 1 16
Enzyme Active Site Is Deeper than Ab Binding
Instead, active site on enzymealso recognizes substrate, butactually complementally fits the transition state and stabilized it.
Ag binding site on Ab binds to Agcomplementally, no further reactionoccurs.
X
Aulani " Biokimia Enzim " Presentasi 1 17
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 18
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 19
Active Site Avoids the Influence of Water
Preventing the influence of water sustains the formation of stable ionic bonds
-+
Aulani " Biokimia Enzim " Presentasi 1 20
Essential of Enzyme Kinetics
E S+ P+
Steady State Theory
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.
SE E
Aulani " Biokimia Enzim " Presentasi 1 21
Constant ES Concentration at Steady State
S P
EES
Reaction Time
Con
cen
tratio
n
Aulani " Biokimia Enzim " Presentasi 1 22
The “Active” EnzymeExamine the hydrolysis of an ester:
O
ORR'
+ H2OR
O
OH
HO R'+very slow
Weak electrophile Poor nucleophile
Expected transition state
O
ORR'
O
H H
O
ORR'
O
H H
Aulani " Biokimia Enzim " Presentasi 1 23
The “Active” EnzymeBase catalyzed hydrolysis of an ester:
O
ORR'
O-
H
O
OHR
O
O-R
HO R'+ +-O R'
Catalysis is accelerated by altering the poor nucleophile H2O into a strong nucleophile OH-
Aulani " Biokimia Enzim " Presentasi 1 24
The “Active” EnzymeAcid catalyzed hydrolysis of an ester:
Catalysis is accelerated by altering the weak electrophile C into a strong nucleophile C+
O
ORR'
+ H3O+
+OH
ORR' C+
OH
ORR'
C
OH
ORR'
O
H H
O
OHR
HO R'+
Aulani " Biokimia Enzim " Presentasi 1 25
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 26
The “Active” EnzymeEnzyme catalyzed hydrolysis of an ester:
Active site incorporates both:
• a base [-B:]
• an acid [-B+-H]
O
ORR'
O
H HB:
B+
H
-O
HB+
B:
H
C
OH
ORR'
C
O
R
O
HB+
B:
H
HO R'
O
ORR'
O
H HB:
B+
H
Aulani " Biokimia Enzim " Presentasi 1 27
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 28
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 29
An Example for Enzyme Kinetics (Invertase)
Vmax
Km S
vo
1/S
1vo
Double reciprocal Direct plot
1)1) Use predefined amount of Enzyme → E
2)2) Add substrate in various concentrations → S (x
3)3) Measure Product in fixed Time (P/t) → vo (y
4)4) (x, y) plot get hyperbolic curve, estimate → Vmax
5)5) When y = 1/2 Vmax calculate x ([S]) → Km
1Vmax
- 1 Km
1/2
Aulani " Biokimia Enzim " Presentasi 1 30
A Real Example for Enzyme KineticsD
ata
no
1234
0.250.501.02.0
0.420.720.800.92
Absorbance v (mole/min)[S]
0.210.360.400.46
(1) The product was measured by spectroscopy at 600 nm for 0.05 per mole(2) Reaction time was 10 min
VelocitySubstrate Product Double reciprocal
1/S 1/v421
0.5
2.081.561.351.16
→→ → →
1.0
0.5
0
v
Dire
ct p
lot
Dou
ble
reci
proc
al 2.0
1.0
0
1/v
-4 -2 0 2 41/[S]
0 1 2[S]
1.0
-3.8
Aulani " Biokimia Enzim " Presentasi 1 31
Enzyme Inhibition (Mechanism)
I
I
S
S
S I
I
I II
S
Competitive Non-competitive Uncompetitive
EE
Different siteCompete for
active siteInhibitor
Substrate
Ca
rtoo
n G
uid
eEq
uatio
n an
d De
scrip
tion
[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].
[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.
[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].
E + S → ES → E + P + II↓EII
←
↑
E + S → ES → E + P + + II II↓ ↓EII + S →EIIS
←
↑ ↑
E + S → ES → E + P + II ↓ EIIS
←
↑
EI
S X
Aulani " Biokimia Enzim " Presentasi 1 32
Km
Enzyme Inhibition (Plots)
I II Competitive Non-competitive Uncompetitive
Dir
ect
Plo
tsD
ou
ble
Rec
ipro
cal
Vmax Vmax
Km Km’ [S], mM
vo
[S], mM
vo
II II
Km [S], mM
Vmax
II
Km’
Vmax’Vmax’
Vmax unchangedKm increased
Vmax decreasedKm unchanged
Both Vmax & Km decreased
II
1/[S]1/Km
1/vo
1/ Vmax
II
Two parallellines
II
Intersect at X axis
1/vo
1/ Vmax
1/[S]1/Km 1/[S]1/Km
1/ Vmax
1/vo
Intersect at Y axis
= Km’
Aulani " Biokimia Enzim " Presentasi 1 33
Ser195
His 57
Asp 102
H–O–CH2
O
C–O -
=
Active Ser
H–N N
C C
C
H
H
CH2
Ser195
His 57
Asp 102
- O–CH2
OC–O–H
=
N N–H
C C
C
H
H
CH2
Aulani " Biokimia Enzim " Presentasi 1 34
pH Influences Chymotrypsin Activity
5 6 7 8 9 10 11
pH
Relative
Activity
Aulani " Biokimia Enzim " Presentasi 1 35
pH
Influ
en
ces N
et C
harg
e o
f Pro
tein
+Net Charge of a Protein
Buffer pH
Isoelectric point,pI
-
3
4
5
6
7
8
9
10
0+
Aulani " Biokimia Enzim " Presentasi 1 36
Imidazole on Histidine Is Affected by pH
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–CH2
O
C–O -
=
H–N N–H
C C-H
C
CH2
H
Aulani " Biokimia Enzim " Presentasi 1 37
Chymotrypsin Produces New Ile16 N-Terminal
I16L13 Y146
Asp 194
–CH2COO-
Ile 16NH2–
Ile 16+ NH3–
5 6 7 8 9 10 11pH
Relative activity
pH 9 pH 10pKa
New NH2-terminus
Aulani " Biokimia Enzim " Presentasi 1 38
New Ile16 N-Terminal Stabilizes Asp194
Asp 102
His 57 Ser 195
Asp 194
Gly 193
Ile 16
+NH3
Catalytic Triad
Aulani " Biokimia Enzim " Presentasi 1 39
O (CH3)2CH–O– P –O–CH(CH3)2
F
=
Chymotrypsin Ser195 Inhibited by DIFP
Diisopropyl-fluorophosphate (DIFP)
O-…H
CH2
Ser 195
O (CH3)2CH–O– P –O–CH(CH3)2
=
O
CH2
Ser 195
XXXX
Aulani " Biokimia Enzim " Presentasi 1 40
Addition of Substrate Blocks DIFP Inhibition
Reaction time
Percent Inhibition of activity (%
)
100
50
0
No substrate
Add substrate
S
+ DIFP
+ DIFP & substrate
XXXX
Aulani " Biokimia Enzim " Presentasi 1 41
Chymotrypsin Also Catalyzes Acetate
O-C N- H
O-C O-
Peptide bond
Ester bond
O
CH3–C–O– –NO2
Nitrophenol acetate
HO– –NO2
O
CH3–C–OH
Hartley & Kilby
Chymotrypsin+ H2O
Nitrophenol
Acetate
No acetate was detected at early stage
Aulani " Biokimia Enzim " Presentasi 1 42
O -
C
Time (sec)N
itrop
hen
ol
Two-Stage Catalysis of Chymotrypsin
O
CH3–C–O– –NO2
Nitrophenol acetate
OC
O
CH3–C HO– –NO2
+ H2O
O-HC
CH3COOH
Kinetics of reaction
Two-phasereaction
Acylation
Deacylation (slow step)
Aulani " Biokimia Enzim " Presentasi 1 43
Extra Negative Charge Was Neutralized
O-C N- H
O-C-OH
NH2-
-C-C-N-C-C-N-C-C-N- H H
E + S
O -
-C N- HO H
O -
-C N- HO H
Aulani " Biokimia Enzim " Presentasi 1 44
Active Site Stabilizes Transition State
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
Specificity Site
Active Site
Aulani " Biokimia Enzim " Presentasi 1 45
xRegulatory
subunit
o
Regulation of Enzyme Activity
o xS I
x oS
Sx
S
oS
AA
Po R xR
+
III
or
inhibitor
proteolysis
phosphorylation
cAMP orcalmodulin
or
regulatoreffector
P
(-)
(+)
Inhibitor Proteolysis
Phosophorylation
Signal transduction
Feedback regulation
Aulani " Biokimia Enzim " Presentasi 1 46
Classification of Proteases
MetalProtease
SerineProtease
CysteineProtease
AspartylProtease
Carboxy-peptidase A
ChymotrypsinTrypsin
Papain
PepsinRenin
H57H57
D102D102
S195-OS195-O--
C25-SC25-S--
H195H195
D215D215
D32D32H2O
Non-specific
Non-specific
AromaticBasic
Non-polar
EDTAEGTA
DFPTLCKTPCK
PCMBLeupeptin
Pepstatin
Family Example Mechanism Specificity Inhibitor
E72E72 H69H69
Zn2+
H196H196
Aulani " Biokimia Enzim " Presentasi 1 47
Serine Protease and AchEChymotrypsin – Gly – Asp – Ser – Gly – Gly – Pro – Leu – Trypsin – Gly – Asp – Ser – Gly – Gly – Pro – Val – Elastase – Gly – Asp – Ser – Gly – Gly – Pro – Leu –Thrombin – Gly – Asp – Ser – Gly – Gly – Pro – Phe –Plasmin – Gly – Asp – Ser – Gly – Gly – Pro – Leu –Acetylcholinesterase – Gly – Glu – Ser – Ala – Gly – Gly – Ala –
Chymotrypsin – Val – Thr – Ala – Ala – His – Cys – Gly – Trypsin – Val – Ser – Ala – Gly – His – Cys – Tyr – Elastase – Leu – Thr – Ala – Ala – His – Cys – Ile – Thrombin – Leu – Thr – Ala – Ala – His – Cys – Leu – Plasmin – Leu – Thr – Ala – Ala – His – Cys – Leu – Acetylcholinesterase – – – – – – – – – – – – – His – – – – – – – –
Ser
19
5
Chymotrypsin – Thr – Ile – Asn – Asn – Asp – Ile – Thr –Trypsin – Tyr – Leu – Asn – Asn – Asp – Ile – Met – Elastase – Ser – Lys – Gly – Asn – Asp – Ile – Ala – Thrombin – Asn – Leu – Asp – Arg – Asp – Ile – Ala – Plasmin – Phe – Thr – Arg – Lys – Asp – Ile – Ala – Acetylcholinesterase – – – – – – – – – – – – – – Asp – – – – – – –
His
57
Asp
10
2
Aulani " Biokimia Enzim " Presentasi 1 48
Sig
moid
al C
urv
e E
ffect
Sigmoidal curve
Exaggeration of sigmoidal curveyields a drastic zigzag line that shows the On/Off point clearly
Positive effector (ATP)brings sigmoidal curveback to hyperbolic
Negative effector (CTP)keeps
Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity
Noncooperative(Hyperbolic)
Cooperative(Sigmoidal)
CTPATP
vo
vo
[Substrate]Off On
Aulani " Biokimia Enzim " Presentasi 1 49
T
T
R
T
[S]
vo
Mechanism and Example of Allosteric Effect
S S
R
R
SS
RS
A
IT
[S]
vo
[S]
vo
(+)
(-) X X
X
R = Relax(active)
T = Tense(inactive)
Allosteric siteHomotropic(+)Concerted
Heterotropic
(+)Sequential
Heterotropic(-)Concerted
Allosteric site
Kinetics CooperationModels
(-)
(+)
(+)
Aulani " Biokimia Enzim " Presentasi 1 50
Activity Regulation of Glycogen Phosphorylase
PA
PA
P
P
A
A
Covalent modificationCovalent modification
P
P
GP kinase
GP phosphatase 1
No
n-co
valent
No
n-co
valent
PA
PA
P
P
PA
PAA
A
A
AMP
ATPGlc-6-PGlucoseCaffeine
GlucoseCaffeine
spontaneously
R
T
R
T
Aulani " Biokimia Enzim " Presentasi 1 51