8/6/2019 Enzyme_revised 2011 Chatchawin
1/111
EnzymeEnzyme
Chatchawin Petchlert, Ph.D.
Department of Biochemistry
Faculty of Science, Burapha University
ChymotrypsinChymotrypsin
8/6/2019 Enzyme_revised 2011 Chatchawin
2/111
OUTLINEOUTLINE
xGENERAL PROPERTIES
xCOFACTORS
xCHEMISTRY OF CATALYST
xENZYME KINETICS
xENZYME INHIBITION
xENZYME REGULATIONS
8/6/2019 Enzyme_revised 2011 Chatchawin
3/111
3
Binding of asubstrate to anenzyme at the activesite.The enzymechymotrypsin, withbound substrate inred (PDB ID 7GCH).Some key active-siteamino acid residuesappear as a redsplotch on theenzyme surface.
8/6/2019 Enzyme_revised 2011 Chatchawin
4/111
4
EnzymeEnzyme
Enzyme-substrate complex
Ribozyme
Abzyme
8/6/2019 Enzyme_revised 2011 Chatchawin
5/111
5
History of EnzymologyHistory of Enzymology
Kuhne, 1878 Enzyme inyeastBuchner, 1897
Sumner, 1926 urease Jackbean Moore Stein, 1963 (aminoacid sequence)
ribonucleasePhillips, 1965 (three
dimensional structure) l soz me
8/6/2019 Enzyme_revised 2011 Chatchawin
6/111
6
8/6/2019 Enzyme_revised 2011 Chatchawin
7/111
GENERAL PROPERTIESGENERAL PROPERTIES
t DEFINITION
Enzyme : a biological catalyst with
*small amount
** G Keq *high efficiency and high specificity
*Almost all enzymes are globular proteins.
8/6/2019 Enzyme_revised 2011 Chatchawin
8/111
8
10-5 M
10-4 10-3 M
Turnover number (S)
(P)
8/6/2019 Enzyme_revised 2011 Chatchawin
9/111
9
S P k = rateconstant equilibrium k1 = 10-3 min-1, k-1 = 10-5 min-1
vf = k1 [S] = vr = k-1 [P]Keq = = = = 100
k1 k-1 106
K
eq= = = =
100
k1
k-1
[P][S] k1k-110
-3
10-5
[P][S]
k1k-1
10310
(Free energy change, G) (Equilibrium constant, Keq)
8/6/2019 Enzyme_revised 2011 Chatchawin
10/111
10
(High specificity)
2 - (substrate
specificity)- (reaction
specificity)
- (chemical catalyst)
8/6/2019 Enzyme_revised 2011 Chatchawin
11/111
veravera
reactionreactionE (enzyme) +S (substrate) Step I :
Binding
ES(enzyme-substratecomplex)
Step II :Transformation
E +P roduct
8/6/2019 Enzyme_revised 2011 Chatchawin
12/111
t SPECIFICITY OF ACTIONSPECIFICITY OF ACTION
1. Absolute specificity E S
2. Relative specificity E S Proteolytic enzyme
t ENZYME NOMENCLATUREENZYME NOMENCLATURE
Trivial namerefer reaction and/or substrate specificity
Hexokinase, glucose phosphotransferaseSystematic name 4 (E.C. number)
1.4.3.4 Monoamine : O
2oxidoreductase
The 1st number : ClassThe 2nd figure : Subclass
The 3rd figure : Sub-subclassThe 4th figure : Serial number of the enzyme in the sub-
subclass
8/6/2019 Enzyme_revised 2011 Chatchawin
13/111
13
6 oxidoreductase dehydrogenase, transferase, hydrolase, lyase,isomerase ligase 3 (trivial name) (systematicname)
1 : 2 ase lactatepyruvate NAD+ 2
8/6/2019 Enzyme_revised 2011 Chatchawin
14/111
14
8/6/2019 Enzyme_revised 2011 Chatchawin
15/111
15
6
1 Oxidoreductase (oxidation-reduction) (redox reaction)
Aox + Bred Ared + Box
2 Transferase
8/6/2019 Enzyme_revised 2011 Chatchawin
16/111
16
()
3 Hydrolase (hydrolysis)
AB + H2O AOH + BH
4 Lyase
ABC AB + C
8/6/2019 Enzyme_revised 2011 Chatchawin
17/111
17
()
5 Isomerase
ABC BAC
6 Ligase 2
ATP
8/6/2019 Enzyme_revised 2011 Chatchawin
18/111
COFACTORCOFACTOR : Non-protein substances for optimum activitywith loosely bound or covalently bound
PROSTHETIC GROUPPROSTHETIC GROUP : cofactor when covalently bound,become a permanent part of enzyme molecule
INORGANIC IONSINORGANIC IONS : Zn2+
, Mg2+
, Mn2+
, Fe2+
COENZYMECOENZYME : organic substance - water soluble vitamin
COFACTORSCOFACTORS
8/6/2019 Enzyme_revised 2011 Chatchawin
19/111
COFACTORSCOFACTORS
HOLOENZYME(Protein~cofactor)(optimally active catalyst)
ease ofdissociation is variable
Protein Cofactor(apoenzyme ;inactive or less
active)
(inorganic ion or
organic substance ;
inactive as a catalyst)
8/6/2019 Enzyme_revised 2011 Chatchawin
20/111
8/6/2019 Enzyme_revised 2011 Chatchawin
21/111
8/6/2019 Enzyme_revised 2011 Chatchawin
22/111
tACTIVE SITEACTIVE SITE
EnzymeEnzyme ACTIVE SITE Binding site Catalytic site
Binding site : substrate
ionic bond, H-bond, Van de Waals forces,hydrophobic interaction
Catalytic site : binding site
8/6/2019 Enzyme_revised 2011 Chatchawin
23/111
t ACTIVE SITEACTIVE SITE
1. Active site
2. Active site 3
3. Substrate active site non-covalent bonding active site
substrate
4. active site substrate
active site
substrate St S ifi it (O t 1948)
8/6/2019 Enzyme_revised 2011 Chatchawin
24/111
Stereo Specificity (Ogston, 1948)
B
C
D
B
C
DBC
D
These two triangles are
not identical
A
The tetrahedral structure
of carbon orbital has rigid
steric strain which makes
the basic building unit of
protein conformation
Juang RH (2004) BCbasics
sp3
Enzyme surface
Three point attachmentThree point attachment 3 3
8/6/2019 Enzyme_revised 2011 Chatchawin
25/111
t ENZYME SPECIFICITYENZYME SPECIFICITY
Model enzyme substrate
1. Lock and Key Model - Fischer (1890) E-S 2. Induced Fit Model - Koshland (1958) E change
3. Strain or Transition State Stabilization Model
- Haldane (1930), Pauling (1948) S change
8/6/2019 Enzyme_revised 2011 Chatchawin
26/111
Induced fit modelInduced fit model
E i ti 2 t iti
8/6/2019 Enzyme_revised 2011 Chatchawin
27/111
Paulings Hypothesis
I think that enzymes are molecules that arecomplementary in structure to the activated complexes of
the reactions that they catalyse, that is, to the molecular
configuration that is intermediate between the reacting
substances and the products of reaction for these
catalysed processes.
The attraction of the enzyme molecule for the activated
complex would thus lead to a decrease in its energy andhence to a decrease in the energy of activation and to an
increase in the rate of reaction. Linus PaulingNature161, 707 (1948)
Enzymes in action 2: transition
state stabilisation
(Binding energy)
(strain) (Distortion)
27
Th N t f E C t l i
8/6/2019 Enzyme_revised 2011 Chatchawin
28/111
The Nature of Enzyme Catalysis
Enzyme provides a catalytic surfaceEnzyme provides a catalytic surface This surface stabilizes transition stateThis surface stabilizes transition state
Transformed transition state to productTransformed transition state to product
B
BA Catalytic surface
A
Juang RH (2004) BCbasics
E St bili T iti St t
8/6/2019 Enzyme_revised 2011 Chatchawin
29/111
Enzyme Stabilizes Transition State
S
P
ES
EST
EP
ST
Reaction direction
Energy change
Energyrequired(nocatalysis)
Energydecr
eases(undercatalysis)
Whats the difference?T = Transition state
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
A ti Sit I D B i d P k t
8/6/2019 Enzyme_revised 2011 Chatchawin
30/111
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
+
-
Juang RH (2004) BCbasics
Stickase
8/6/2019 Enzyme_revised 2011 Chatchawin
31/111
Stickase
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 stateAdapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
X
Enzyme Active Site Is Deeper than Ab Binding
8/6/2019 Enzyme_revised 2011 Chatchawin
32/111
Enzyme Active Site Is Deeper than Ab Binding
Instead, active site on enzyme
also recognizes substrate, butactually complementally fits thtransition state and stabilized it
binding site on Ab binds to Agplementally, no further reaction
urs.
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
X
Active Site Avoids the Influence of Water
8/6/2019 Enzyme_revised 2011 Chatchawin
33/111
Active Site Avoids the Influence of Water
g the influence of water sustains the formation of stable io
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115
-+
8/6/2019 Enzyme_revised 2011 Chatchawin
34/111
Enzyme KineticsEnzyme Kinetics
Enzyme Kinetics
8/6/2019 Enzyme_revised 2011 Chatchawin
35/111
Enzyme Kinetics
Increase the substrate concentration,
observe the change of enzyme activity
Substrate concentrationExam Chapters
Scor
e
Enzy
mea
cti vity
Student A
Student B
Student C
0 1 2 3 4 0 1 2 3 4
Juang RH (2004) BCbasics
8/6/2019 Enzyme_revised 2011 Chatchawin
36/111
36
Invertase (IT)
8/6/2019 Enzyme_revised 2011 Chatchawin
37/111
Invertase (IT)
ITSucrose
Non-reducing sugarReducing
sugars
Glucose Fructose
Reducing Power
+
HOCH2
O
OH
1
2
34
5
66
5
4
32
1
1
2
3
4
5
6
HOCH2
O
OH
O
HOCH2 HOCH2
OH
H2O
O
HOCH2HOCH2
HO
O
HOCH2
OHOCH2HOCH2
O
CHO
H-C-OHHO-C-H
H-C-OH
H-C-OH
H2-C-OH
H2C-OH
C=OHO-C-H
H-C-OH
H-C-OH
H2-C-OHJuan
gRH
( 2004)BCbasics
1 2
In
8/6/2019 Enzyme_revised 2011 Chatchawin
38/111
ncreaseSubstr
ateConc
entration
21 3 4 5 6 7 80
0 2 4 6 8
Substrate mole
Product
80
60
40
20
0
S
+E
P(inafixedperiod
oftJuang RH (2004) BCbasics
Essential of Enzyme Kinetics
8/6/2019 Enzyme_revised 2011 Chatchawin
39/111
Essential of Enzyme Kinetics
E S+ P+
Steady State TheorySteady 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
Juang RH (2004) BCbasics
Constant ES Concentration at Steady State
8/6/2019 Enzyme_revised 2011 Chatchawin
40/111
Constant ES Concentration at Steady State
S P
EES
Reaction Time
Con
centra
tion
Juang RH (2004) BCbasics
An Example for Enzyme Kinetics (Invertase)
8/6/2019 Enzyme_revised 2011 Chatchawin
41/111
An Example for Enzyme Kinetics (Invertase)
Vmax
Km S
vo
1/S
1vo
Double reciprocal Direct plot
1)1)Use predefined amount ofEnzyme E
2)2)Add substratein various concentrations S (x)3)3)Measure Productin fixed Time (P/t) vo(y)
4)4)(x, y)plot get hyperbolic curve, estimateVmax
5)5)Wheny = 1/2 Vmax calculate x ([S]) Km
1
Vmax
- 1
Km
1/2
Juan
gRH
( 2004)BCbasics
A Real Example for Enzyme Kinetics
8/6/2019 Enzyme_revised 2011 Chatchawin
42/111
A Real Example for Enzyme Kinetics
Data
no
12
3
4
0.250.50
1.0
2.0
0.420.72
0.80
0.92
Absorbancev (mole/min)[S]0.210.36
0.40
0.46
(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/v
42
1
0.5
2.081.56
1.35
1.16
1.0
0.5
0
v
Directplot
Doublerec
iproca
l2.0
1.0
0
1/v
-4 -2 0 2 41/[S]0 1 2 [S]
1.0
-3.8
Juan
gRH
( 2004)BCbasics
Enzyme Kinetics
8/6/2019 Enzyme_revised 2011 Chatchawin
43/111
Enzyme Kinetics
vo=
Vmax [S]
Km+[S]
KmVmax &
E1E2E3
1st order
zero order
Competitive
Non-competitive
Uncompetitive
Direct plot
Double reciprocal
Bi-substrate reaction alsofollows M-M equation, butone of the substrate shouldbe saturated when estimatethe other
Affinity with
substrate
Maximum
velocity Inhibition
Activity
Observe vo change
under various [S],resulted plotsyield Vmax andKm
k3 [Et]
kcatTurn overnumber
kcat /Km
Activity Unit
1molemin
Specific Activity
unitmg
Significance
Juang RH (2004) BCbasics
Significance of Enzyme Kinetics
8/6/2019 Enzyme_revised 2011 Chatchawin
44/111
Significance of Enzyme Kinetics
vo = Vmax [S]
Km+[S]ObtainVmax andKm
[S] = Low High [S] = Fixed concentration
zero order
1st order
E3
E2E1
Proportio
nalto
enzymeconcen
tration
v0 = Vmax K = k3 [Et] K
Juang RH (2004) BCbasics
K : Affinity with Substrate
8/6/2019 Enzyme_revised 2011 Chatchawin
45/111
Km= [S]
Km+[S] = 2[S]
Vmax2
=Vmax [S]Km+[S]
Km: Affinity with Substrate
Ifvo =Vmax
2
S2S1 S3
S1S2 S3
Vmax
1/2
When using different substrate
Affinity changesKm
vo =
Vmax [S]
Km+[S]
Juan
gRH
( 2004)BCbasics
K : Hexokinase Example
8/6/2019 Enzyme_revised 2011 Chatchawin
46/111
Km: Hexokinase Example
Glucose + ATP Glc-6-P + ADP
1
2
3
4
5
6
Glucose Allose MannoseSubstratnumber
Km
= 8 8,000 5 M
CHO
H-C-OHHO-C-H
H-C-OH
H-C-OH
H2-C-OH
CHO
H-C-OH
H-C-OH
H-C-OH
H-C-OH
H2-C-OH
CHOHO-C-HHO-C-H
H-C-OH
H-C-OH
H2-C-OH
Juang RH (2004) BCbasics
8/6/2019 Enzyme_revised 2011 Chatchawin
47/111
Turn Over Numbers of Enzymes
8/6/2019 Enzyme_revised 2011 Chatchawin
48/111
Turn Over Numbers of Enzymes
Catalase H2O2
Carbonic anhydrase HCO3-
Acetylcholinesterase Acetylcholine
40,000,000
400,000
140,000
-Lactamase Benzylpenicillin 2,000Fumarase Fumarate 800
RecA protein (ATPase) ATP 0.4
Enzymes Substrate kcat (s-1
)
The number of product transformed from substrate
by one enzyme molecule in one second
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.263
Chymotrypsin Has Distinct k t /K to
8/6/2019 Enzyme_revised 2011 Chatchawin
49/111
Chymotrypsin Has Distinct kcat /Km to
Different SubstratesO R O
H3CCNCCOCH3
H H
= =
HGlycine
kcat / Km
1.3 10-1
CH2CH2CH3Norvaline 3.6 102
CH2CH2CH2CH3Norleucine 3.0 103
CH2Phenylalanine 1.0 105
(M-1
s-1
)
R =
Adapted from Mathews et al (2000) Biochemistry (3e) p.379
Enzyme Activity Unit
8/6/2019 Enzyme_revised 2011 Chatchawin
50/111
Enzyme Activity Unit
Reaction time(min)
P
roduct[P
]
0 10 20 30 40
Slope
tan
S Pmole
vo = [P]/min
Unit =
Activity Units
Protein (mg)
t
mole
/min
y
x
y
x
= tan
Juan
gRH
( 2004)BCbasics
SpecificActivity =
Enzyme Inhibition (Mechanism)
8/6/2019 Enzyme_revised 2011 Chatchawin
51/111
Enzyme Inhibition (Mechanism)
I
I
S
S
S II
I II
S
CompetitiveNon-competitiveUncompetitiv
EE
Different siteCompete foractive siteInhibitor
Substrate
Cartoo
nGuide
E
quat
ion
and
Des
cription
[II] binds to free [E] only,
and competes with [S];
increasing [S] overcomes
Inhibition by [II].
[II] binds to free [E] or [ES]
complex; Increasing [S] can
not overcome [II] inhibition.
[II] binds to [ES] complex
only, increasing [S] favors
the inhibition by [II].
E + SESE + P
+II
EII
E + SESE + P
+ +II II
EII+SEIIS
E + SESE + P
+ II
EIIS
E
I
S X
Juang RH (2004) BCbasics
Enzyme Inhibition (Plots)
8/6/2019 Enzyme_revised 2011 Chatchawin
52/111
Km
Enzyme Inhibition (Plots)
I IICompetitiveNon-competitiveUncompetitiv
D
irect
Plots
Double
Rec
iprocal
Vmax Vmax
Km Km [S], mM
vo
[S], mM
vo
II II
Km [S], mM
Vmax
II
Km
Vmax Vmax
Vmax unchanged
Km increased
Vmax decreased
Km unchangedBoth Vmax & Km decreased
II
1/[S]1/Km
1/vo
1/Vmax
II
Two parallellines
II
Intersectat X axis
1/vo
1/Vmax
1/[S]1/Km 1/[S]1/Km
1/Vmax
1/vo
Intersectat Y axis
=Km
Juang RH (2004) BCbasics
Competitive Inhibition
8/6/2019 Enzyme_revised 2011 Chatchawin
53/111
Competitive Inhibition
Succinate Glutarate Malonate Oxalate
Succinate Dehydrogenase
Substrate Competitive InhibitorProduct
Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49
C-OO-
C-HC-H
C-OO-
C-OO-
H-C-HH-C-H
C-OO-
C-OO-
H-C-HH-C-H
H-C-H
C-OO-
C-OO-
C-OO-
C-OO-
H-C-H
C-OO-
Sulfa Drug Is Competitive Inhibitor
8/6/2019 Enzyme_revised 2011 Chatchawin
54/111
Sulfa Drug Is Competitive Inhibitor
-COOHH2N-
-SONH2H2N-
Precursor Folicacid Tetrahydro-folic acid
Sulfanilamide
Sulfa drug (anti-inflammation)
Para-aminobenzoic acid (PABA)
Bacteria needs PABA for
the biosynthesis of folic acid
Sulfa drugs has similar
structure with PABA, andinhibit bacteria growth.
Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.197
Domagk (1939)
Enzyme Inhibitors Are Extensively Used
8/6/2019 Enzyme_revised 2011 Chatchawin
55/111
Enzyme Inhibitors Are Extensively Used
Sulfa drug (anti-inflammation)
Pseudo substratePseudo substrate competitive inhibitor
Protease inhibitorPlaques in brain contains protein inhibitor
HIV protease is critical to life cycle of HIV
HIV proteaseHIV protease(homodimer):(homodimer):
inhibitor is used to treat AIDS Symmetr
Notsymmetr
Human aspartyl protease:(monodimer)
domain 1
Asp Asp
domain 2
subunit 2
Asp
subunit 1
Asp
Juang RH (2004) BCbasics
Alzheimer's disease
HIV protease vs Aspartyl protease
8/6/2019 Enzyme_revised 2011 Chatchawin
56/111
p p y p
Asymmetrimonomer
HIV proteaseHIV protease (homodimer)
HIV Proteaseinhibitoris used in treating AIDS
Symmetricdimer
Asp
subunit 2
Aspartyl protease (monomer)
subunit 1
Asp
domain 1 domain 2
Asp Asp
Juang RH (2004) BCbasics
8/6/2019 Enzyme_revised 2011 Chatchawin
57/111
Enzyme CatalysisEnzyme Catalysis
Chymotrypsin Catalytic Mechanism A1
8/6/2019 Enzyme_revised 2011 Chatchawin
58/111
Asp102
His57
Ser195
Catalytic TriadCatalytic Triad
HH
y yp y
N
C
C
N
[HOOC]H
O
C
C
N
C
C
[NH2]
CC
O
Check substrate specificity
8/6/2019 Enzyme_revised 2011 Chatchawin
59/111
Chymotrypsin Catalytic Mechanism A3
8/6/2019 Enzyme_revised 2011 Chatchawin
60/111
HH
y yp y
N
C
C
N
[HOOC]H O
C
C
N CC
[NH2]CC
O
Acyl-Enzyme Intermediate
Chymotrypsin Catalytic Mechanism D1
8/6/2019 Enzyme_revised 2011 Chatchawin
61/111
H
y yp y
N-H
C
C
N
[HOOC]H
O
C
C
N CC
[NH2]CC
O
HO
H
Acyl-Enzyme Water Intermediate
Chymotrypsin Catalytic Mechanism D2
8/6/2019 Enzyme_revised 2011 Chatchawin
62/111
H
y yp y
O
O
C
C
N CC
[NH2]CC
H
Second Transition State
OH
8/6/2019 Enzyme_revised 2011 Chatchawin
63/111
Chymotrypsin Is Activated by Proteolysis
8/6/2019 Enzyme_revised 2011 Chatchawin
64/111
y yp y y
Ada
ptedfro
m
Campbell(19
99)B
iochemistr
y
(3d)p
.179
245
R15-I16
Chymotrypsinogen (inactive)
-Chymotrypsin(active)
S14-R15 T147-N148
Trypsin
-Chymotrypsinactive
-Chymotrypsin
I16L13 A149Y146
Disulfide bonds
8/6/2019 Enzyme_revised 2011 Chatchawin
65/111
pH Influences Chymotrypsin Activity
8/6/2019 Enzyme_revised 2011 Chatchawin
66/111
5 6 7 8 9 10 11
pH
RelativeAc
tivity
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162
8/6/2019 Enzyme_revised 2011 Chatchawin
67/111
8/6/2019 Enzyme_revised 2011 Chatchawin
68/111
8/6/2019 Enzyme_revised 2011 Chatchawin
69/111
8/6/2019 Enzyme_revised 2011 Chatchawin
70/111
Chymotrypsin Ser195 Inhibited by DIFP
8/6/2019 Enzyme_revised 2011 Chatchawin
71/111
O
(CH3)2CHOPOCH(CH3)2
F
=
Diisopropyl-fluorophosphate (DIFP)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167
O-H
CH2
Ser 195
O
(CH3)2CHOPOCH(CH3)2
=
O
CH2
Ser 195
XXXX
8/6/2019 Enzyme_revised 2011 Chatchawin
72/111
Chymotrypsin Also Catalyzes Acetate
8/6/2019 Enzyme_revised 2011 Chatchawin
73/111
O
-C N-
H
O
-C O-
Peptide bond
Ester bond
OCH3CO NO2
Nitrophenol acetate
HO NO2
O
CH3COH
Hartley & Kilby
Chymotrypsin+ H2O
Nitrophenol
Acetate
No acetate was detected at early stage
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168
Two-Stage Catalysis of Chymotrypsin
8/6/2019 Enzyme_revised 2011 Chatchawin
74/111
O-
C
Time (sec)
Nitrophenol
O
CH3CO NO2
Nitrophenol acetate
O
C
O
CH3C HO NO2
+ H2O
O-HC
CH3COOH
Kineticsofreactio
n
Two-phasereaction
Acylation
Deacylation (slow step)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169
8/6/2019 Enzyme_revised 2011 Chatchawin
75/111
Active Site Stabilizes Transition State
8/6/2019 Enzyme_revised 2011 Chatchawin
76/111
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
Ada
ptedfrom
Dressle
r&Po
tter
(1991)Discovering
Enzymes
,p.197
8/6/2019 Enzyme_revised 2011 Chatchawin
77/111
Basic Mechanism of Catalysis
8/6/2019 Enzyme_revised 2011 Chatchawin
78/111
3 basic types 1) Bond Strain
2) Acid-base transfer3) Orientation
Conformational chan
Chemical reaction
Space arrangement
Carboxypeptidase ACarboxypeptidase BCarboxypeptidase Y
Concert
equentialChymotrypsinTrypsinElastase
non-polarRK
non-specific
YFWRK
GA
Ser-proteaseEndopeptidase
Metal proteaseExopeptidase
Juang RH (2004) BCbasics
Concerted Mechanism of Catalysis
8/6/2019 Enzyme_revised 2011 Chatchawin
79/111
1
2
3 4
5O
-
H+
H
COO-
(270)Glu
(248)Tyr
O-
H
His(196)
His (69)
Glu(72)
+Arg (145)
Carboxypeptidase A
C-terminus
ACTIVESITE
ACTIVESITE
Check for
C-terminal
Site forspecificit
Activesitepocket
Substrate
peptidechain
RNCN C
COO-O-
C
+Zn
J
uangRH
( 2004)BCbas
ics
Chymotrypsin Has A Site for Specificity
8/6/2019 Enzyme_revised 2011 Chatchawin
80/111
O ONCCNCC NCCNCC
R H R
O-
C
Ser
Active SiteActive Site
Specificity
Site
Specificity
Site Catalytic Site
Juang RH (2004) BCbasics
Specificity of Ser-Protease Family
8/6/2019 Enzyme_revised 2011 Chatchawin
81/111
COO-
C
Asp
COO-
C
Asp
Active Site
Trypsin Chymotrypsin Elastase
cut at Lys, Arg cut at Trp, Phe, Tyr cut at Ala, Gly
Non-polarpocket
Deepand
ne
gativel y
charge
dpocket
Shallow andnon-polar
O O
CNCCN
C
CC
C
NH3
+
O O
CNCCN
C
O O
CNCCN
CH3
Jua
ngRH
( 2004)BCbasics
Control Points of Gene Regulation
8/6/2019 Enzyme_revised 2011 Chatchawin
82/111
Prokaryotics
DNA
ribosomemRNA
proteins
Post-translationalcontrol
Eukaryotics
proteins
cap5 3
tail
maturemRNA
DNA
53process
mRNA
Juang RH (2004) BCbasics
Translation
Activity
Proteolysis
TranscriptionRNA Processing
RNA Transport
RNA Degradation
Regulation of Enzyme Activity
8/6/2019 Enzyme_revised 2011 Chatchawin
83/111
xRegulatory
subunit
o
o x
S I
x o
S
S
x
S
o
S
AA
Po R x
R
+
I
II
or
inhibitor
proteolysis
phosphorylation
cAMP orcalmodulin
or
regulatoreffector
P
(-)
(+)
Inhibitor Proteolysis
Phosophorylation
ignal transduction
eedback regulation
Jua
ngRH
( 2004)BCbasics
Cascade Amplification of Signals
8/6/2019 Enzyme_revised 2011 Chatchawin
84/111
Cascade
nS nP1 Enzyme
Juang RH (2004) BCbasics
8/6/2019 Enzyme_revised 2011 Chatchawin
85/111
85
8/6/2019 Enzyme_revised 2011 Chatchawin
86/111
86
8/6/2019 Enzyme_revised 2011 Chatchawin
87/111
8/6/2019 Enzyme_revised 2011 Chatchawin
88/111
88
8/6/2019 Enzyme_revised 2011 Chatchawin
89/111
8/6/2019 Enzyme_revised 2011 Chatchawin
90/111
90
8/6/2019 Enzyme_revised 2011 Chatchawin
91/111
91
Two views of the regulatory enzyme aspartate trans-carbamoylase (derived from PDB ID 2AT2) The regulatory
8/6/2019 Enzyme_revised 2011 Chatchawin
92/111
92
carbamoylase (derived from PDB ID 2AT2). The regulatoryclusters form the points of a triangle surrounding the
catalytic subunits. Binding sites for allosteric modulatorsare on the regulatory subunits. Modulator binding produces
large changes in enzyme conformation and activity.
8/6/2019 Enzyme_revised 2011 Chatchawin
93/111
Phosphorylation
Fischer Kreb (1978)
8/6/2019 Enzyme_revised 2011 Chatchawin
94/111
Conformational
ChangedephosphorylastionPhosphatase
P
Protein
OH
SerSerThr Tyr(His)
Active Inactive
Inactive Active
Glycogen phosphorylase b Glycogen phosphorylase a
Fischer, Kreb (1978)
Juang RH (2004) BCbasics
Kinasephosphorylation
Regulation of Blood Sugar
Cori & Cori (1947)
8/6/2019 Enzyme_revised 2011 Chatchawin
95/111
gh blood sugargh blood sugarInsulin
Pancreas Glycogen Glucose
Decrease
Glycogen
synthase
IncreaseIncrease
Hormone
Signal TransductionSignal Transduction
Blood
LiverLiver
w blood sugarGlucagon
GTP-protein-linked receptor
Tyrosine-kinase-linked receptor
Glycogen
phosphorylase
Cori & Cori (1947)
Juang RH (2004) BCbasics
8/6/2019 Enzyme_revised 2011 Chatchawin
96/111
Glycogen Phosphorylase, GP
8/6/2019 Enzyme_revised 2011 Chatchawin
97/111
Glycogen
n n-1
Glc-1-P Glc-6-P Glycolysis
Glycogenphosphorylase a*
Ph
osp
ha
ta
se
GPkinase
Glycogenphosphorylase b
(inactive)
ATP
Proteinkinase A
cAMP
Phosphorylase
+
+
AMP (+)
ATP (-)
Glc-6-P (-)
Glucose (-)
Caffeine (-)
P
RT6.2
6.3
6.4
P
P
Juang RH (2004) BCbasics
8/6/2019 Enzyme_revised 2011 Chatchawin
98/111
cAMP Is the Second Messenger
GlSutherland (1971)
8/6/2019 Enzyme_revised 2011 Chatchawin
99/111
G protein
Glycogen Glc-1-P
PGP1 aGP b
PGP KinaseGP Kinase
Protein Kinase AProtein Kinase A
cAMPATP
CyclaseG protein
Adenylate cyclase
A Cyclic AMP (second messenger)
GP kinase
GP
ReceptorGlucagonSutherland (1971)
Cascade
Inactive
Active
JuangRH
( 2004)BCbasics
Receptors on Cell Membrane
G protein linked ReceptorGilman Rodbell (1994)
8/6/2019 Enzyme_revised 2011 Chatchawin
100/111
SH2domain
G protein
GDP
+ Signal
-GDP
+GTP
GDP
GTP
GTP
Adenylate cyclase
+ Signal
ActivationP
ProteinPhosphatase
GlycogenSynthase
GlycogenSynthase
P
active
Insulin
P P
PP kinase
Glucagon
A
G-protein-linked Receptor
Enzyme-linked ReceptorThe third group:Ion-channel-linked Receptor
Gilman, Rodbell (1994)
Glycogen breadkdown
Glycogen synthesis
JuangRH
( 2004)BCbasics
GP kinase Phosphatase
8/6/2019 Enzyme_revised 2011 Chatchawin
101/111
P
P
A
GP kinase
GP a
GP b
Glycogen synthase
Glycogen synthase P
Protein phosphatase-1
Protein phosphatase-1
Protein phosphatase inhibitor-1
Protein phosphatase inhibitor-1
Glycogen
PKA
P
active
inactive
Glu
cago
n
Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.217
Signal Transduction
8/6/2019 Enzyme_revised 2011 Chatchawin
102/111
Receptor
Hormone Signal
G
Cyclase
Transducer
Effector Enzyme
Effector
Effect
G-protein
Juang RH (2004) BCbasics
Allosteric Enzyme ATCase
Active relaxed form
8/6/2019 Enzyme_revised 2011 Chatchawin
103/111
CCC
+
Active relaxed form
Inactive tense form
ATCase
RR
RR
RR
CCC
COO-
CH2HN-C-COO-
H H
-
--
-O
H2N-C-O-PO32-=
OH2N-C-
=
COO-
CH2N-C-COO-
H H
-
--
-
Catalytic subunits
Catalytic subunits
Regulatory subunits
ATP
CTP
Nucleic acid
metabolism
Feedback
inhibition
AspartateCarbamoylphosphate
Carbamoyl aspartate
CTP
CTP
CTP
CTP
CTP
CTP
Juang RH (2004) BCbasics
Quaternary structure
Sigm
Positive effector
Noncooperative
(Hyperbolic)
vo
8/6/2019 Enzyme_revised 2011 Chatchawin
104/111
moid
alCurve
Effect
Sigmoidal curve
Exaggeration ofsigmoidal curveyields a drastic
zigzag line thatshows the On/Offpoint clearly
Positive effector
(ATP)
brings sigmoidal
curveback to hyperbolic
Negative effector
(CTP)
keeps
Consequently,
Allosteric enzymecan sense theconcentration ofthe environment andadjust its activity
Cooperative
(Sigmoidal)
CTPATP
vo
[Substrate]
Off On
Juang RH (2004) BCbasics
Mechanism and Example of Allosteric Effect
8/6/2019 Enzyme_revised 2011 Chatchawin
105/111
T
T
R
T
[S]
voS
S
R
R
SS
R
S
A
I
T[S]
vo
[S]
vo
(+)
(-) X X
X
R = Relax(active)
T = Tense
(inactive)
Allosteric site
Homotropic(+)
Concerted
Heterotropic
(+)
Sequential
Heterotropic
(-)
Concerted
Allosteric site
Kinetics CooperationModels
(-)
(+)
(+)
Juang RH (2004) BCbasics
Activity Regulation of Glycogen Phosphorylase
8/6/2019 Enzyme_revised 2011 Chatchawin
106/111
P
A
PA
P
P
A
A
Covalent modificationCovalent modification
P
P
GP kinase
GP phosphatase 1
Non-covalent
Non-cov
alent
P
A
PA
P
PPA
PAA
A
A
AMP
ATP
Glc-6-P
Glucose
Caffeine
Glucose
Caffeine
spontaneo
usly
R
T
R
T
Ga
rrett&G
risham
(1999)Bioche
mistry(2e)
p.679
Major Metabolic PathwayGlycolysis
STAGE 1
8/6/2019 Enzyme_revised 2011 Chatchawin
107/111
Citricacidcycle
P
P
P 2
1
$ NADH$$
ATPH3-C-H
H3-C-OH
H2-C=O
H-COOH
O=C=O
HH4
3
2
1
0
Mitochondria
Kinase
Oxidative phosphorylation
Glycolysis
A
AcetylCoA
Pyruvate Pyruvate
STAGE 1
Macromolecule Unit molecule
STAGE 2 Unit molecule Key small molecule
STAGE 3 Energy production
H2O
CO2
6
3
3
Change
of carbonnumber
xidation of Carbon
Glc-6-P GlycogenGlc-1-PGlycogen phosphorylase
OO0
1
1
2
2
Glucose
Starch
digestion
JuangRH
( 2004)BCbasic
s
Operon Expression Regulated by Its Metabolites
8/6/2019 Enzyme_revised 2011 Chatchawin
108/111
ROperator Gene
S S
mRNA
R
S
RNA
Polymerase
Operator Gene
R
RNA
Polymerase
R
P
P
Upstream metabolite (S) inactivates
repressor, and induces the expression
Downstream metabolite (P)might bind and activate repressor,Then turns off the gene expression
X
ON
OFF
R
S
Juang RH (2004) BCbasics
Cross Talk between Cells
Direct contact Diffusion
8/6/2019 Enzyme_revised 2011 Chatchawin
109/111
(R)
Blood
(R)
(S) (R)
Synaptic Endocrine
Local paracrineDirect contact
Neuron impulse
Direct contact Diffusion
S
hortranged
Longranged
Signaling cell (S) Receptor cell (R)
(S)
(S)
Ad
aptedfro
m
Albertsetal
(2002
)MolecularBiolog
yof
theCell(4
e)p.833
How
ShapeSi
1 3 4 6 7 8 9 10 11 122 5
8/6/2019 Enzyme_revised 2011 Chatchawin
110/111
wto
Separa
teThes
eObjects
1 2 3
9 10 11 12
6
4 85
7
4
5
8
wood stone cotton wood wood cotton stone wood stone cotton stone c
cotton
wood
stone
SizeDensity
Shape
Density
Size
Sieving different sizes Different sedimentationDifferent rolling speed
4 6 7 85
1 3 4 6 7 8 9 10 11 122 5
Juang RH (2004) BCbasics
Basic Principles of Protein Purification
8/6/2019 Enzyme_revised 2011 Chatchawin
111/111
Ammonium sulfate fractionation
Cell OrganelleHomogenization
Macromolecule
Nucleicacid
Carbohydrate (Lipid)
Size Charge Polarity Affinity
Small moleculeCell
DebrisProteinAmino acid,
Sugar,
Nucleotides,etc
Gel filtration,SDS-PAGE,
Ion exchange,Chromatofocusing,
Disc PAGE
Reverse phasechromatography,
HIC
Affinitychromatography,
Top Related