enz 2007b.pdf
TRANSCRIPT
-
E1-1
11 E1 22 E2 33 E3 44 E4 55 E5 66 E6 77 E7 88
EnzymeEnzyme
-
E1-2
Phlegm
Blood
YellowBile
BlackBile
Milestones of Science (2000) p.36, National Geographic Society; photo from The Granger Collection, NY
(acetylcholinesterase)
12 3
-
E1-3
Dis
cove
ring
Enzy
me
(199
1) p
.22
-
E1-4
1926 Sumner urease
Juang RH (2007) BCbasics
(glycolysis)
-
E1-5
Sumner
Sumner
Urease crystal Discov
erin
g En
zym
e (1
991)
p.8
2
1930 Sumner urease
Sumner Willsttter Sumner urease urease urease urease
Urease NH2-CO-NH2 + H2O 2NH3 + CO2
-
E1-6
P+
Juang RH (2007) BCbasics
-
E1-7
1
FeCl3
Hemoglobin
Catalase
1,000,000,000
1,000,000
1,000
2H2O2 2H2O + O2
P
ES
Juang RH (2007) BCbasics
( 1 ) catalase catalase Catalase Catalase
-
E1-8
Alberts et al (2002) Molecular Biology of the Cell (4e) p.107
A B
()
(protein-protein interaction) (cross-talk)
-
E1-9
F6P
F1,6bP
PEP
Pyruvate
Ada
pted
from
Buc
hana
n et
al.
(200
0) B
ioch
emis
try &
Mol
ecul
ar B
iolo
gy o
f Pla
nts
p.66
1
A B C
X
Y
Z
+
-
F6P
F1,6bP
PEP
Pyruvate
Animals Plants
ATP-dependentphosphofructokinase
Pyruvate kinase
GlycolysisGlycolysis
( Z) Z ( A)
(12 Biosignaling)
-
E1-10
()
( helix, sheet)
(monomer)
(dimer)
Domain ( )
Juang RH (2007) BCbasics
-
E1-11
Ribozyme -
RNA ( DNA )
RNARNA Tetrahymena (ciliated protozoan) rRNA intron intron (no protein !)
??? WHYWHY ??? intron ()
WC pair (?)
Altman, Cech (1989)
Juang RH (2007) BCbasics; Wikipedia (Tetrahymena)
RNA RNA ribozyme
rRNA intron rRNA intron intron guanosine
RNA
-
E1-12
Ribozyme
Alberts et al (2002) Molecular Biology of the Cell (4e) p.369
viroid RNA RNA RNA ribozyme ribozyme
-
E1-13
Alb
erts
et a
l(20
02) M
olec
ular
Bio
logy
of t
he C
ell (
4e) p
.304
, 347
RN
A
tRNA
rRNA
DNA RNA Watson-Crick W-C RNA RNA RNA ribozyme
Ribozyme RNA RNA
-
E1-14
1
Trypsin Pepsin Renin Lysozyme
-ase
ECEC 4.1.1.22 4 Main Class Lyase1 Subclass C-C lyase1 Sub-subclass carboxylase22 Series number 22
Histidine carboxylase
Enzyme Commission
Juang RH (2007) BCbasics
() () () () RNA
-in zyme (IUBMB) (Enzyme Commission, EC)
-
E2-1
2.1
Apo-enzymeApo-
enzyme Cofactor
Coenzyme
Holoenzyme
Juang RH (2007) BCbasics
(holoenzyme) ()
( chymotrypsinogen chymotrypsin insulin)
-
E2-2
Ada
pted
from
Cam
pbel
l (19
99) B
ioch
emis
try (
3d) p
.179
245
R15-I16
Chymotrypsinogen (inactive)
-Chymotrypsin (active)
S14-R15 T147-N148
Trypsin
-Chymotrypsin (active)
-Chymotrypsin
I16L13 A149Y146
Disulfide bonds
(chymotrypsin)
Chymotrypsin
-
E2-3
Adapted from Stryer (1995) Biochemistry (4e) p.592
AP
AP
4 nm
Active site
Phosphorylation site
AMP siteGlycogen-binding
site
Pyridoxalphosphate site
Glycogen phosphorylase
chymotrypsin (glycogen phosphorylase, GP) chymotrypsin GP
GP GP 180 120 (3603)
-
E2-4
Bio
-Lab
Cel
l 83
(4),
1995
-
-+
+ +
S
S
() ()
-
E2-5
1. Coenzyme [Cofactor]Carboxypeptidase ZnHexokinase ATP
2. Dehydrogenase NADH
3. Pyruvate Vit. B1 Zndecarboxylase (thiamine)
Juang RH (2007) BCbasics
(3)(1) (2)
NADH
-
E2-6
NC CC
NC
H3 C
NH2
=
=
=
CH2
-OP
OPO
CH2CH2
O =
O =
O-
O-
H3CC =N+
C
CS=
-
Thiazolium ring
H3CC =N+
C
CS=
OH
CCH3H
Adapted from Alberts et al (1994) Molecular Biology of the Cell (3e) p.130
B1 thiazolium
-
E2-7
Glu CysHis
2+
Stry
er(1
995)
Bio
chem
istry
(4e)
p.5
92
Carboxypeptidase A
Juan
g R
H (2
008)
BC
basi
cs
(d)
-
E2-8
Thiamine (thiamine pyrophosphate)B1
Riboflavin (FADH)B2 H-
Niacin (NADH, NADPH)B3 H-
Pyridoxine (pyridoxal phosphate)B6 Biotin CO2 Lipoic acid (lipoamine) Folic acid (tetrahydrofolate) CobalaminB12
ATP UDP-Glc Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.170
Todd (1957)
Pantothenic acid (coenzyme A) B5
-
E2-9
NADHbindingdomain
Substratebindingdomain
NADH
Gly-3-P
Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.25
Glyceraldehyde-3-phosphate dehydrogenase
See also:LehningerPrinciples of Biochemistry (4e)p.514, F13-16
Gly-3-P dehydrogenase domains Gly-3-P NADH NADH domain NADH
-
E2-10
NADH
NAD+/NADH 340 nm
240 280 320 360 400
Wave length (nm)
NADH
NAD+
340 nm
NAD+ NADH 340 nm
Dehydrogenase ()Glyceraldehyde-3-P deHase
NADH NADPH
NAD+ domain ()
Juang RH (2007) BCbasics
NAD+ NADH
-
E2-11
NADH
Glyceraldehyde 3-phosphate
NADPH
NADP+
Ada
pted
from
Alb
erts
et a
l (20
02) M
olec
ular
Bio
logy
of t
he C
ell (
4e) p
.86
A B
H2C-OH
OH-C-H
H2C- P
H2C=O
OH-C-H
H2C- P
C=O
HCOH
HCHC
CC
NAD+-NADH A NADH B
-
E2-12
Nicotinamide Adenine Dinucleotide (Phosphate)
HHCONH2
N
PO
P ORibose
P ORibose
Adenine
Ada
pted
from
Alb
erts
et a
l (20
02) M
olec
ular
Bio
logy
of t
he C
ell (
4e) p
.86
NADP+ NADPH
PO
P ORibose
P ORibose
H
CONH2+N
H
Adenine
Nicotinamide
NADH NADH () NAD+ nicotinamide hydride NADH ATP
NADP+ NADPH (2 ) NADP+-NADPH NAD+-NADH (NAD+-NADH)
-
E2-13
hydride hydrogen proton
-
H1.008
1
hydride
+
1s-
-
+-
+-
Juang RH (2007) BCbasics
( hydride, :H-) hydride (H+)
hydride NADH hydride
-
E2-14
Juan
g R
H (2
007)
BC
basi
cs
Base
Sugar
Acid
MonophosphateDiphosphateTriphosphate
AdenineGuanineThymineCytosineUracil
Nucleoside (Adenosine)Nucleotide (Adenosine monophosphate, AMP)
Purine
Pyrimidine
Ribose,Deoxyribose
1
23
4
5
(ribose) (deoxyribose)DNA RNA () RNA (ATP)RNA world
-
E2-15
RNA WorldRNA WorldRNA
RN
A
(1) Ribozyme
(2) RNA
(3) (ATP, NADH) RNA
Juang RH (2007) BCbasics
RNA DNA
-
E2-16
RNA vs DNA
OH
H
Uracil
Thymidine
Juan
g R
H (2
008)
BC
basi
cs
PO42-
OH
()
()
RNA RNA DNA RNA
DNA RNA DNA RNA U T ()DNA
RNA RNA (A=U, CG) RNA RNA RNA DNA DNA RNA (Central dogma)DNA RNA Protein RNA
-
E2-17
Starr (1987) Biology (4e) 548
RNA RNA DNA RNA DNA DNAmRNA
-
E2-18
-
E3-1
11 E1 22 E2 33 E3 44 E4 55 E5 66 E6 77 E7 88
EnzymeEnzyme
() () ()
() () () () () () () ()
3.1 [E] [S] [ES] 3.2 [ES] Michaelis-Menten Vmax Km 3.3
-
E3-2
Stickase
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
X
T
Lehninger Stickase
Stickase (T )
-
E3-3
3.1
B
BA
A
Juang RH (2007) BCbasics
()
(abzyme)
-
E3-4
S
P
ES
EST
EP
ST
T = Transition state
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
-
E3-5
Mathews et al (2000) Biochemistry (3e) p.246
+
(affinity)
(affinity)
E3-2 Stickase
-
E3-6
Lerner & Tramontano (1988) Scientific American (March) p.43
abzyme
Sci Am March: 43, 1988
-
E3-7
NH2 COOH1 NH2 COOH2
NH2 C N COOHO
H21
H2O:
C NO
HC NO
HH2O
T
Juang RH (2008) BCbasics
sp3sp2
(T)
-
E3-8
AbzymeAbzyme
peptide bond
: sp2 [sp3]* sp2
Why ?Why ?Abzyme
sp3
sp3
Juang RH (2007) BCbasics
peptide bond -C-N- sp2 peptide bond sp2 C-OH sp3 sp3
albumin
Nature 383: 23 (1996)
- -
-
E3-9
Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
() sp3
-
E3-10
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
X
() ()
abzyme
-
E3-11
Chymotrypsin
Nel
son
& C
ox(1
993)
Leh
ning
er P
rinci
ples
of B
ioch
emis
try (2
e)
peptidase abzyme
( E5-27)
-
E3-12
(1)
(2)
(3)
(4)
(2)
(3)(4)
(1)
+
-
Juang RH (2007) BCbasics
(1)
(2)
(3) ( Ser-OH) ( His H+)( Ser-O-)
pH ( E5-2)
(4)
-
E3-13
Hexokinase
Alberts et al (1994) Molecular Biology of the Cell (3e) p.196
Hexokinase
domain domains
-
E3-14
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115
-+
()
-
E3-15
3
0 1 2 3 4 0 1 2 3 4
Juang RH (2007) BCbasics
() () (1~3 ) ( 4 ) ()
( 1~2 )
-
E3-16
Michaelis Menten
Michaelis Menten
Nel
son
& C
ox(2
000)
Leh
ning
er P
rinci
ples
of B
ioch
emis
try (3
e) p
.258
(Fischer, Brown & Henri)
Michaelis defensive enzyme defensive enzyme Michaelis-Menten
-
E3-17
Invertase (IT)
ITSucrose
Glucose Fructose
Reducing Power
+HOCH2
O
OH
12
34
566
54
32
1
1
2
3
4
5
6
HOCH2O
OH
OHOCH2 HOCH2
OH
H2O
O
HOCH2HOCH2
HO
O
HOCH2O
HOCH2HOCH2O
CHOH-C-OH
HO-C-HH-C-OHH-C-OH
H2-C-OH
H2C-OHC=O
HO-C-HH-C-OHH-C-OH
H2-C-OH
Juan
g R
H (2
007)
BC
basi
cs
1 2
Michaelis Menten invertase () ()
(E + S ES)
-
E3-18
21 3 4 5 6 7 80
0 2 4 6 8 mole
80
60
40
20
0
S+E
P
(
)
Juang RH (2007) BCbasics
()
-
E3-19
E S+ P+
Steady State TheorySteady State Theory
ES [ES]
SE E
Juang RH (2007) BCbasics
Michaelis Menten [E] [S] [ES] [P][ES] [ES] Steady state theory [ES]
-
E3-20
Steady State ES
S P
EES
Juang RH (2007) BCbasics
ES ES pre-steady state
-
E3-21
E + S ES E + P(vo)k2k1 k3
k1[E][S] = k2 [ES] + k3 [ES]
vo=Vmax [S]Km + [S]
[Et]= [Ef] + [ES]vo = k3 [ES]
Vmax = k3 [Et]
3.1
3.2
ES
[ES] 3.2.1Steady State
Michaelis-Menten
3.2.2
Juang RH (2007) BCbasics
[ES]
-
E3-22
(vo)
3.2.2
k1 [E][S] = k2 [ES] + k3 [ES]
[Et] = [Ef] + [ES]vo = k3 [ES]
Vmax = k3 [Et]
(I)
(II)
(III)
(IV)
E + S ES E + Pk2
k1 k3
Juang RH (2007) BCbasics
Steady state theory [ES] [ES] [ES] [E] [S] k1 [ES] k1 [E][S][ES] [E] + [P] k3 k3 [ES] [E] + [S] k2[ES] [ES] (I)
[Et] [Ef] [ES] (II) [E]
vo [ES] [E] + [P] vo k3 [ES] (III)
[Et] [ES] Vmax(IV) Vmax = k3 [Et]
[ES] (I) Michaelis-Menten
-
E3-23
(1) (I) (k2k3) [ES] = k1 [E][S]
k1 k2k3 [E][S] [ES] = [E][S] Km [ES] = k2k3 k1 Kmvo vo [E][S] k3 [E][S](2) (III) [ES] = vo (V)k3 k3 Km Km
(3) (II) [Ef] [Et] - [ES] [Ef] [E][E] [Et] - [ES] (V)
k3 ([Et]-[ES])[S] k3 [Et][S] - k3 [ES][S]vo Km Km(4) (III) vo k3 [ES] (IV) Vmax k3 [Et]
Vmax [S] - vo [S]vo vo Km Vmax [S] - vo [S] Km vo Km + vo [S] Vmax [S] (VI)
Vmax [S] (5) (VI) vo M-M vo Km + [S]
[ES]
Km
[ES]vo
[E]
Vmax vo
vo
Juan
g R
H (2
007)
BC
basi
cs
y =bx
a + x
Km
-
E3-24
Lineweaver-Burk Plot
(invertase)
Vmax
Km S
vo
1/S
1vo
1)1) (invertase) E2)2) () S (x )3)3) (P/t) vo (y )4)4) (x, y) Vmax5)5) y = 1/2 Vmax x ( [S]) Km
1Vmax
- 1 Km
1/2
Juan
g R
H (2
007)
BC
basi
cs
invertase Michaelis Menten
-
E3-25
1234
0.250.501.02.0
0.420.720.800.92
v (mole/min)[S]0.210.360.400.46
(1) 0.05 1 mole ()(2) 10 min (3) ([S] = 0)
1/S 1/v421
0.5
2.081.561.351.16
1.0
0.5
0
v
2.0
1.0
0
1/v
-4 -2 0 2 41/[S]
0 1 2 [S]
1.0
-3.8
Juan
g R
H (2
007)
BC
basi
cs
( 0) ([S] = 0) (blank)
-
E3-26
vo=Vmax [S]Km + [S]
KmVmax &
3.3
E1E2E3
1st order
zero order
Competitive
Non-competitive
Uncompetitive
M-M
4
3.2.4
3.2.4.1
3.2.4.2
3.2.4.3
3.2.4.4
[S] vo Vmax Km
k3 [Et]
kcatTurn overnumber
kcat /Km
Activity Unit1 mole
min
unitmg
3.2.3
Juang RH (2007) BCbasics
-
E3-27
3.2.3
vo =Vmax [S]Km + [S]
Vmax Km
[S] = [S] =
zero order
1st order
E3E2E1
v0 = Vmax K = k3 [Et] K
Juang RH (2007) BCbasics
M-M Vmax Km M-M
() () () (10 Km)
-
E3-28
Km = [S]
Km + [S] = 2[S]
Vmax2
=Vmax [S]Km + [S]
3.2.4.2 Km
vo =Vmax
2
S2S1 S3
S1 S2 S3
Vmax
1/2
S1, S2, S3
Km
vo =Vmax [S]Km + [S]
Juan
g R
H (2
007)
BC
basi
cs
Km Vmax Km ()
Km Km
-
E3-29
Km : Hexokinase
Glucose + ATP Glc-6-P + ADP
123456
Glucose Allose Mannose
Km = 8 8,000 5 M
CHOH-C-OH
HO-C-HH-C-OHH-C-OH
H2-C-OH
CHOH-C-OHH-C-OHH-C-OHH-C-OH
H2-C-OH
CHOHO-C-HHO-C-H
H-C-OHH-C-OH
H2-C-OH
Juang RH (2007) BCbasics
Hexokinase ( glucose, allose, mannose ) Km Hexokinase glucose mannose ( Km ) allose (Km 8,000 M) -OH hexokinase glucose
hexokinase hexokinase
-
E3-30
3.2.4.3 Turn Over Number, kcat
vo =Vmax [S]Km + [S]
(vo)E + S ES E + P
k2
k1 k3
k3 kcat turn over number (t.o.n.)
=k3 [E][S]Km + [S]
=Km
k3 [E][S]
M-M
(IV)
Juang RH (2007) BCbasics
M-M [S] Km vo = Vmax (III) vo = k3 [ES] (IV) Vmax = k3 [Et] [ES] = [Et] [ES] ES E + P k3 k3 kcat turn over number (t.o.n.) molecular activity
Km vo = (k3/Km) [E][S] [E] [S] k3/Km (kcat/Km) (k3 Km)
-
E3-31
Turn Over Number
Catalase H2O2Carbonic anhydrase HCO3-
Acetylcholinesterase Acetylcholine
40,000,000
400,000
140,000
-Lactamase Benzylpenicillin 2,000
Fumarase Fumarate 800
RecA protein (ATPase) ATP 0.4
kcat (s-1)
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.263
k3 turn over number (t.o.n.) s-1 RecA ATPase t.o.n. 0.4 2.5 ATP ADP (DNA )
-
E3-32
O R O H3CCNCCOCH3
H H
= =
Chymotrypsin kcat /Km
HGlycine
kcat / Km1.3 10-1
CH2CH2CH3Norvaline 3.6 102
CH2CH2CH2CH3Norleucine 3.0 103
CH2Phenylalanine 1.0 105
(M-1s-1)
R =
Adapted from Mathews et al (2000) Biochemistry (3e) p.379
kcat/Km Vmax (kcat)chymotrypsin
kcat/Km 10 8~10 9 (M-1s-1) triose phosphate isomerase 2.410 8
-
E3-33
3.2.4.4
(min)
[P]
0 10 20 30 40
tan
S P mole
vo = [P] / min
Unit =
=
tt
mole/min
yx
yx = tan
Juan
g R
H (2
007)
BC
basi
cs
mole 1 mole (U) mg (U/mg)
30 min ( tan ) 40 min ()
30 min 1 min
-
E3-34
3.3
S1 + S2 P
S P1 + P2
S1 + S2 P1 + P2
(Bi-Uni, -)
(Uni-Bi,-)
(Bi-Bi, -)
Glucose + ATP Glc-6-P + ADPHexokinase
M-M
Juang RH (2007) BCbasics
hexokinase ATP Glc-6-P ADP
hexokinase
Gonick, L. & Wheelis, M. The Cartoon Guide to Genetics,
-
E4-1
11 E1 22 E2 33 E3 44 E4 55 E5 66 E6 77 E7 88
EnzymeEnzyme
-
E4-2
Juang RH (2007) BCbasics
-
E4-3
4
Competitive
Non-competitive
Uncompetitive
Penicillin
(Hg, Pb)
DFP, TPCK Sarin (-Ser) PCMB (-Cys)
(Mixed inhibition)
Juang RH (2007) BCbasics
() ()
-
E4-4
()
I
I
S
S
S I
I
I II
S
Competitive Non-competitive Uncompetitive
EE
[II] [E] [S] [S] [II]
[II] [E] [S] [ES] [S] [II]
[II] [ES] [S] [II]
E + SESE + P+II
EII
E + SESE + P+ +II II
EII+ SEIIS
E + SESE + P+II
EIIS
EI
S X
Juang RH (2007) BCbasics
(competitive) non-competitive uncompetitive competitive
-
E4-5
Km
()
I II Competitive Non-competitive Uncompetitive
Vmax Vmax
Km Km [S], mM
vo
[S], mM
voII II
Km [S], mM
Vmax
II
Km
VmaxVmax
Vmax Km Vmax Km Vmax Km
II
1/[S]-1/Km
1/vo
1/Vmax
II
II
X
1/vo
1/Vmax
1/[S]-1/Km 1/[S]-1/Km
1/Vmax
1/vo
Y
= Km
Juang RH (2007) BCbasics
-
E4-6
Succinate Glutarate Malonate Oxalate
Succinate Dehydrogenase
Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49
C-OO-
C-H
C-H
C-OO-
C-OO-
H-C-H
H-C-H
C-OO-
C-OO-
H-C-H
H-C-H
H-C-H
C-OO-
C-OO-
C-OO-C-OO-
H-C-H
C-OO-
-
E4-7
-COOHH2N-
-SONH2H2N-
FolicacidTetrahydro-
folic acid
Sulfanilamide ()
Para-aminobenzoic acid (PABA)
PABA
PABA
Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.197
Domagk (1939)
PABAPABA
-
E4-8
Sulfa drug ()Pseudo substrate Pseudo substrate
Protease inhibitor (Alzheimer's disease)
HIV protease HIV proteaseHIV protease ((homodimerhomodimer):):
inhibitor AIDS
aspartyl protease:(monodimer)
domain 1
Asp Asp
domain 2
subunit 2
Asp
subunit 1
Asp
Juang RH (2007) BCbasics
(PABA)PABA
AIDS
-
E4-9
Trypsin
Inhibitor
Mat
hew
s et
al (
2000
) Bio
chem
istry
(3e)
p.1
99
Stryer (1995) Biochemistry (4e) p.252
-
E4-10
Alzheimer
Dressler & Potter (1991) Discovering Enzymes, p.245
Amyloid -peptide Aggregation
Protease Protease (ACE)(ACE)
Hu et al (2001) J Biol Chem 276:47863-47868
-
E4-11
X
Nowak and McMichael (1995) Scientific American, no. 8, p. 42
HIV (human immune deficiency virus) RNA DNA DNA RNA
-
E4-12
gag pol envmRNA
translation
protein (gag-pol)
HIV protease
p17 p11 (protease)
p15
p24
p32 (intergrase)
p66/51 (reverse transcriptase)
Adapted from Garrett & Grisham (1999) Biochemistry (2e) p.522C
ampb
ell (
1999
) Bio
chem
istry
(3d
) p.3
29
HIV
-
E4-13
HIV protease vs Aspartyl protease
HIV protease HIV protease (homodimer)HIV Protease inhibitor HIV
Asp
subunit 2
Aspartyl protease (monomer)
subunit 1Asp
domain 1 domain 2
Asp Asp
Juang RH (2007) BCbasics
HIV aspartyl protease Asp () HIV aspartyl protease HIV HIV protease HIV protease
proteases domain subunit HIV protease Asp protease domains Asp domains
-
E4-14
Asp Protease Asp
Stryer (1995) Biochemistry (4e) p.228
Aspartyl protease Asp pepsin () pH 2~3 Asp
domains HIV protease
-
E4-15
Asp
Asp
Nat
ure
(199
6) 3
84S
p.2
5
HIV protease HIV protease aspartyl protease
-
E4-16
HIV Protease
Stryer (1995) Biochemistry (4e) p.230
Drug design
Combinatory Chemistry
HIV Protease
HIV Proteaseinhibitor
Asp
(combinatory chemistry)
-
E4-17
GT
PenicillinPenicillin
[E]-Cys-SH ... PCMB protease inhibitor
GT Ser-OH () GT D-Ala-D-Ala ()
[E]-Ser-OH ... DFP, TPCK ( chymotrypsin); TLCK (trypsin) SarinSarin ( acetylcholinesterase)
Fleming (1945)
Juang RH (2008) BCbasics
cysteine protease Cysserine protease Ser ( Cys, Ser) cysteine protease PCMB Cys
-
E4-18
GlycopeptideTranspeptidase
(GT)
Stryer (1995) Biochemistry (4e) p.201
tetrapeptide
pentaglycine
Sugar chain
(glycopeptide transpeptidase, GT) GT GT
-
E4-19
Penicillin GT
PenicillinPenicillin
OHSer
Glycopeptidetranspeptidase
(inactive)
OH
HC C C
O=C
HN C=OR
N CH
S CH3CH3COO-
H
O Ser
Glycopeptidetranspeptidase
Adapted from Stryer (1995) Biochemistry (4e) p.202
(GT) Ser Ser -OH GT Ser
-
E4-20
Penicillin
PenicillinPenicillin
Adapted from Stryer (1995) Biochemistry (4e) p.203
D-Alanine
D-Alanine
S
GT GT
-
E4-21
COO-ClHg
Para-chloro-mercuribenzoic acid (PCMB)
E -CH2-SHCys Cl-
E -CH2-S COO-Hg
E -CH2-OHSer F-
Diisopropyl-fluorophosphate (DIFP)
Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.200
OCH(CH3)2F P= O
OCH(CH3)2
E -CH2-OOCH(CH3)2
P = O
OCH(CH3)2
-SH Cys -OH Ser
Ser Cys
-
E4-22
Ada
pted
from
Dre
ssle
r & P
otte
r(20
00) D
isco
verin
g En
zym
es,p
.249
Juan
g R
H (2
007)
BC
basi
cs
acetylcholinesterase SarinSer
-
E5-1
11 E1 22 E2 33 E3 44 E4 55 E5 66 E6 77 E7 88
EnzymeEnzyme
(5.1) (5.2, 5.3) Ser protease chymotrypsin (5.3) Ser protease (5.4) (6.1)
-
E5-2
HO
H
Adapted from Nelson & Cox
(2000) Lehninger Principles of
Biochemistry (3e) p.252
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 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
( Fast )
-
E5-3
5.1
1) Bond Strain2) Acid-base transfer3) Orientation
Carboxypeptidase ACarboxypeptidase BCarboxypeptidase Y
ChymotrypsinTrypsinElastase
non-polarRK
non-specific
YFWRKGA
Ser-
Juang RH (2007) BCbasics
() ()
carboxypeptidase (CP) chymotrypsin (trypsin) CP chymotrypsin
-
E5-4
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-
RNCN C
COO-O-
C
+Zn
Juan
g R
H (2
007)
BC
basi
cs
carboxypeptidase
(1) Zn2+ carbonyl (2)
(3) Glu270 OH- C+ (2) C-OH
(4) Tyr 248-OH lone pair
(5) C- R Arg145 C- -COOH C-
-
E5-5
Carboxypeptidase A
Stryer (1995) Biochemistry (4e) p.220
Arg 145
Tyr 248
Glu 270
Carboxypeptidase A Tyr248
-
E5-6
Stryer (1995) Biochemistry (4e) p.220
- -
-
E5-7
Chymotrypsin
Stry
er(1
995)
Bio
chem
istry
(4e)
p.2
07
Branden & Tooze (1999) Introduction to Protein Structure (2e) p.212, 210
Chymotrypsin chymotrypsinchymotrypsin
-
E5-8
Chymotrypsin
Ada
pted
from
Cam
pbel
l (19
99) B
ioch
emis
try (
3d) p
.179
245
R15-I16
Chymotrypsinogen (inactive)
-Chymotrypsin (active)
S14-R15 T147-N148
Trypsin
-Chymotrypsinogen (active)
-Chymotrypsin
I16L13 A149Y146
Disulfide bonds
Chymotrypsin trypsin R15-I16 Y146-A149 chymotrypsin
chymotrypsin chymotrypsin
-
E5-9
Chymotrypsin
Catalytic triad: Asp102His57Ser195 charge relaycharge relay(1)(1) pH
(2)(2)
(3)(3) -C-O- Gly193 Ser195 -N-H
(4)(4) non-polar pocket
Acylation: N-peptide (Ser195) Deacylation: N-peptide (slow step)
Nitrophenyl acetate ()
His 57 (pKa = 6): pH > 6, imidazole H+ (charged)Ile 16 (new N-terminal): pH > 9, NH3+ H+ ()Ser 195: DIFP Ser-OH
Juang RH (2007) BCbasics
Chymotrypsin Ser protease (catalytic triad)
Catalytic triad Ser195 -OH His57 -O- -O- (carbonyl C+)
chymotrypsin
(1) pH (2) (3) (4)
chymotrypsin Scientific American Library Discovering enzymes (by D. Dressler & H. Potter, 1991)
-
E5-10
Chym
otrypsin
Ser195
His 57
Asp 102
HOCH2OCO-
=
Active Ser
HN N
C C
C
H
H
CH2
Ser195
His 57
Asp 102
-OCH2OCOH
=
N NH
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
Chymotrypsin (57, 102, 195) Asp102 His57 His57 Ser195 Ser195 () (catalytic triad)
-
E5-11
Chymotrypsin
5 6 7 8 9 10 11
pH
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162
chymotrypsin chymotrypsin pH
-
E5-12
Juan
g R
H (2
007)
BC
basi
cs
+Net Charge of a Protein
Buffer pH
Isoelectric point,pI
-
3456789
10
0+
pH pH (isoelectric point, pI) pI
pH pI ( pI = 6 pH = 9) pH pI pI pI pH
-
E5-13
Histidine imidazole
HN N
C C
C
H
H
H+
pH < 6 pH > 7
+HN NH
C C
C
H
H
Inactive+ Ser
195
His 57
Asp 102
HOCH2OCO-
=
HN NH
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 En
zym
es,p
.163
pH 7 6 chymotrypsin pH Chymotrypsin
pKa pH His imidazole (6.0) pH His pH 6 His Ser195 His Ser195
-
E5-14
Chymotrypsin Ile16 N-
I16L13 Y146
Asp 194
CH2COO-
Ile 16NH2
Ile 16+NH3
5 6 7 8 9 10 11pH
pH 9 pH 10pKa
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.165
New NH2-terminus
pH 9 pKa 9 pKa 10 N- pKa 9
chymotrypsin Ile16 N- NH2 pH 9 Asp194 Asp194 Ser195 His57 pH 10
-
E5-15
Ile16 N- 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
Ile16 N- Asp194 Ser195 His57
-
E5-16
O
(CH3)2CHO POCH(CH3)2F
=
Chymotrypsin Ser195 DIFP
Diisopropyl-fluorophosphate (DIFP)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167
O-HCH2
Ser 195
O
(CH3)2CHO POCH(CH3)2
=
O
CH2
Ser 195
XXX
DIFP Ser Ser195 DIFP chymotrypsin (pseudosubstrate)
-
E5-17
Reaction time
(%
)
100
50
0
S
+ DIFP
+ DIFP & substrate
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167
XXX
DIFP chymotrypsin
-
E5-18
Asp102
His57
Ser195
Catalytic TriadCatalytic Triad
HH
Chymotrypsin A1
NC
CN
[HOOC]H
O
CC
NC
C[NH2]
CC
O
Check substrate specificity
Chymotrypsin
Asp102
His57
Ser195HH
Chymotrypsin A2
NC
CN
[HOOC] HO
CC
NC
C[NH2]
CC
O
First Transition State
HH
Chymotrypsin A3
NC
CN
[HOOC] H O
CC
NC
C[NH2]CC
O
Acyl-Enzyme Intermediate
H
Chymotrypsin D1
N-HC
CN
[HOOC] H
O
CC
NC
C[NH2]CC
O
H OH
Acyl-Enzyme Water Intermediate
H
Chymotrypsin D2
O
O
CC
NC
C[NH2]CC
H
Second Transition State
OH
H
Chymotrypsin D3
O
CC
NC
C[NH2]
CC
O
OH
Deacylation
H
Asp102
His57
Ser195
Catalytic TriadCatalytic Triad
HH
Chymotrypsin A1
NC
CN
[HOOC]H
O
CC
NC
C[NH2]
CC
O
Check substrate specificity
-
E5-19
AbzymeAbzyme
peptide bond
: sp2 [sp3]* sp2
Why ?Why ?
Abzyme
sp3
sp3
Juang RH (2007) BCbasics
-C-N- sp2 sp2 C-OH sp2 sp3 -CO-N- -CO-O- amide
carbonyl (C=O) -O: () Chymotrypsin (1) Ser -O: (2)
-
E5-20
O-C N-
H
O-C O-
Peptide bond
Ester bond
OCH3CO NO2
Nitrophenol acetate
HO NO2
OCH3COH
Hartley & Kilby
Chymotrypsin+ H2O
Nitrophenol
Acetate
acetate Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168
chymotrypsin nitrophenol acetate nitrophenol
nitrophenol acetate nitrophenol acetate nitrophenol acetate acetate
(Hartley & Kilby) chymotrypsin
-
E5-21
O-C
Time (sec)N
itrop
heno
l
Chymotrypsin
OCH3CO NO2
Nitrophenol acetate
OC
OCH3C HO NO2
+ H2OO-HC
CH3COOH
Acylation
Deacylation (slow step)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169
chymotrypsin acylation deacylation nitrophenol acetate nitrophenol phases nitrophenol
acylation nitrophenol deacylation rate-limiting stepnitrophenol acetate
-
E5-22
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
Chymotrypsin -N-H () Chymotrypsin
-
E5-23
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
Ada
pted
from
Dre
ssle
r & P
otte
r(19
91) D
isco
verin
g En
zym
es,p
.197
Gly193 Ser195 -N-H ( Trp)
-
E5-24
O ONCCNCC NCCNCC
R H R
Chymotrypsin
O-CSer
Juang RH (2007) BCbasics
N- (R)Chymotrypsin Trp, Phe, Tyr
-
E5-25
Trypsin Lys vs Asp (specific binding)
Trypsin chymotrypsin, elastase ...Serine proteaseSerine protease
[Science]
Trypsin (Asp189) Chymotrypsin (Ser189)
Juang RH (2007) BCbasics
Ser catalytic triad Ser195 Ser protease catalytic triad
() (trypsin) (chymotrypsin)
trypsin CHOM trypsin
-
E5-26
Trypsin
COO-CAsp
COO-CAsp
Trypsin Chymotrypsin Elastase Lys, Arg Trp, Phe, Tyr Ala, Gly
Non-polarpocket
Dee
p an
d ne
gativ
ely
char
ged
pock
et Shallow andnon-polar
pocket
O OCNCCN
CCCCNH3+
O OCNCCN
C
O OCNCCN
CH3
Juan
g R
H (2
007)
BC
basi
cs
Ser
Trypsin Asp189 Lys Arg
-
E5-27
TrypsinAsp 189
ChymotrypsinSer 189 Asp
HisSer
Ester (+)Amide (-)
Hedstrom et al (1992) Science 255: p.1250
trypsin Asp189 chymotrypsin Ser amide bond
-
E5-28
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
-
E5-29
5.4.3
BC
DB CDB C
D
A
Juang RH (2007) BCbasics
sp3
sp3 sp3 A, B, C, D (1) (2)
A B, C, D ()
D-, L- L-
-
E5-30
PowerPoint Enz(5A)
-
E5-31
Substrate binding site
+
Lysozyme
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
pH 5
Chair Half-chair
H-bonds
O
O
sp3
sp2
sp2
Lysozyme ()
()D chair half-chair Asp52 ()
carbonium sp2 half-chair D52 -COO-
-COOH ()pH 5 D52 E35
-
E5-32
Catalytic toolkit for active sites
Gutteridge A, Thornton JM (2005) Understanding natures catalytic toolkit. TIBS 30: 622
Combinations of different residues form catalytic unitsthat are found repeatedly in different unrelated enzymes
HistidineHistidineHistidine
Arg-Arg-Arg Asp-Asp (Thr ) Asp-Arg ( pKa) ArgAsp-COO- ()
Asp-Lys-Thr triad (reactive -O-) Asp-Lys ( Tyr pKa) KEK (EK K112 pKa) ()
Arg, carboxlyate
Lysine
Histidine
Asp-His-Ser triad (reactive -O-) Asp-His () His-His dyad (H83 )
Arg COO- (a) Adenylate kinase Arg Asp Arg (b) Cardosin Asp215 Asp32 Thr218 Ser35 Asp pKa(c) Asp180 Arg179 Asp pKa (d) Asp192 Arg190 COO-
Lys (a) Asparaginase triad Asp-Lys Thr95 reactive Thr (b) Asp-Lys Tyr58 O- Tyr pKa(c) Lys-Glu-Lys triad Lys112 Glu51-Lys53 Lys112 pKa(d) Glu-Lys
His (a) Ser-His-Asp triad His-Asp Ser (b) Asp His dyad ()(c) Phosphotransferase His-His dyadHis83 N His68 tautomer Thr86 His 68 tautomer
-
E5-33
Amazing histidine
Gutteridge A, Thornton JM (2005) TIBS 30: 622 WIKIPEDIA
H57
S195
D102
Petsko GA, Ringe D (2004) Protein Structure and Function F4-35
Histidine imidazole ( lone pair ) N-H ()
His Cys Cys Cys ()
His CysSer protease catalytic triad (His) (Asp) (Ser) ()
-
E5-34
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
His (H119) (H12)H12 P His119
()Lys41
H119 shuttle
-
E5-35
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
AICAR transformylase - IMP cyclohydrolase (ATIC)
-
E5-36
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
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 2003 GAPDH (Histone H2B) GAPDH
GAPDH
-
E6-1
11 E1 22 E2 33 E3 44 E4 55 E5 66 E6 77 E7 88
EnzymeEnzyme
() ()
RNA
Lehninger Principles of Biochemistry (12) ()
-
E6-2
DNA
ribosomemRNA
proteins
RNA ProcessingRNA Transport
RNA Degradation
Post-translationalcontrol
proteins
cap5 3
tail
mature mRNA
DNA5
3processmRNA
Juang RH (2007) BCbasics
DNA RNA () mRNA
-
E6-3
x
o
6
o xS I
x oS
Sx
S
oS
AA
Po R xR
+
III
or
inhibitor
proteolysis
phosphorylation
cAMP orcalmodulin
or
regulatoreffector
P
(-)
(+)
4 6.1
6.2
6.3
6.4
Juan
g R
H (2
007)
BC
basi
cs
(6.1) (6.2) cAMP calmodulin (6.3) (6.4)
-
E6-4
1 2 3 4 5 6 7 8 9 10 11
pH
100
50
Activity (%)
00 20 40 60 80 100
Temperature (C)
Pepsin Urease Arginase
Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.47
pH () PCR DNA polymerase Taq polymerase
-
E6-5
6.1
Insulin (): A chain & B chain; C chain
Fibrin Thrombin cascade ()
Trypsin, Chymotrypsin, ElastaseCT N- (Ile16) Asp194 Ser195
ZymogenZymogen ()
morphine vs endophine (YGGFM)
Juang RH (2007) BCbasics
mRNA (zymogen)
-
E6-6
Fibrin
Mat
hew
s et
al (
2000
) Bio
chem
istry
(3e)
p.4
06
thrombin fibrinogen fibrin (factor VIII)
-
E6-7
Mat
hew
s et
al (
2000
) Bio
chem
istry
(3e)
p.4
07
fibrinogen fibrin Ser
-
E6-8
Scientific American (1998) April p.92
(A) (B) (C) (D) (E) (F) (G) (H) (I) (J) (K) (L) (M) (N)
-
E6-9
Cascade
nS nP1 Enzyme
Juang RH (2007) BCbasics
XII
XI
IX
X
T
F
() () ( G protein) ( cyclase)
-
E6-10
Trypsinogen
Chymotrypsinogen
-Chymotrypsin-Chymotrypsin
-Chymotrypsin-Chymotrypsin
TrypsinTrypsin
ProcarboxypeptidaseProelastase
CarboxypeptidaseCarboxypeptidaseElastaseElastase
EnteropeptidaseEnteropeptidase
Adapted from Mathews et al (2000) Biochemistry (3e) p.404
C
T
-
E6-11
Chymotrypsin
I16L13 A149Y146
+NH3
Ser 195
Asp 194
Gly 193
Ada
pted
from
Dre
ssle
r & P
otte
r(19
91) D
isco
verin
g En
zym
es,p
.206
Ile 16
Stryer (1995) Biochemistry (4e) p.222
C T C
chymotrypsin Ile16 N- Asp194 Ser195 Ser
-
E6-12
Alberts et al (2002) Molecular Biology of the Cell (4e) p. 667 Stryer (1995) Biochemistry (4e) p.248
Lysosome
-
E6-13
Enkephalin
Tyr
Gly
GlyPhe
Met
e e e e e e e e
Trypsin-like endo-protease
e
Dressler & Potter (1991) Discovering Enzymes, p.247
trypsin
-
E6-14
MetalProtease
SerineProtease
CysteineProtease
AspartylProtease
Carboxy-peptidase A
ChymotrypsinTrypsin
Papain
PepsinRenin
H57H57
D102D102
S195S195--OO--
C25C25--SS--
H195H195
D215D215
D32D32H2O
Non-specific
Non-specific
AromaticBasic
Non-polar
EDTAEGTA
DFPTLCKTPCK
PCMBLeupeptin
Pepstatin
E72E72 H69H69
Zn2+
H196H196
Juang RH (2007) BCbasics
metal protease Ser Cys protease Ser Cys Asp protease Asp
Ser trypsin chymotrypsin
Ser ()
-
E6-15
Alberts et al (2002) Molecular Biology of the Cell (4e) p. 143
Alb
erts
et a
l(19
94) M
olec
ular
Bio
logy
of t
he C
ell (
3e) p
. 120
similar
deletionidentical
conserved
Ser ( Ser )
-
E6-16
Subtilisin
No enzyme 1
Asn155 Leu 10,000,000(Asn155 )
His & Asp Ala 37,000Ser, His & Asp Ala 4,000Subtilisin 10,000,000,000
Triad: Ser His Asp
Ser Ala 5,000Asp Ala 330,000
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.245
Site-directed mutagenesis
Ser protease subtilisin Ser195 His57 Asp102 Asn155 Leu Ser protease
-
E6-17
Serine AchE
Chymotrypsin 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
Se
r 1
95
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
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.244
Ser Ser ()
acetylcholinesterase (AchE) AchE ()AchE Ser
-
E6-18
O CH3COH
CH3HOCH2CH2NCH3
CH3
++AchE
Acetylcholine
O CH3CH3COCH2CH2NCH3
CH3
+Choline
Dale, Loewi (1936)
Ada
pted
from
Dre
ssle
r & P
otte
r(19
91) D
isco
verin
g En
zym
es,p
.239
Sarin
Acetylcholinesterase (AchE) (acetylcholine)
AchE Ser C=O
Sarin chymotrypsin (Ser) AchE chymotrypsin Sarin AchE
-
E6-19
H
AchE
AchE Ser
O-
CH
O CH3CH3COCH2CH2NCH3
CH3
+
H-O-H
AchE
OC
H
OCH3C
CH3HOCH2CH2NCH3
CH3
+H2O
AchE
O-
CH H
O CH3COH
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.243
AchE
O-
CH
OCH3C
CH3OCH2CH2NCH3H CH3
+
Deacylation
Acylation
Acetylcholinesterase Ser His Asp acylation deacylation
-
E6-20
O-
C
Sesame Triad
Hi, Everybody!Hi, Everybody!
Juang RH (2007) BCbasics
-
E6-21
Divergent evolution
Asp--His--SerAsp--His--Ser
Convergent evolution
TrypsinChymotrypsin Elastase Thrombin Plasmin
AcetylcholinesteraseAcetylcholinAcetylcholinesteraseesterase
ThyroglobulinThyroglobulinThyroglobulin
C NC
C
H
O
CC
CO
O
Ester bond
Peptide bond
EvolutionEvolutionMolecularMolecular
acetylcholine
Serine ProteaseSer
Juang RH (2007) BCbasics
Acetylcholinesterase (AchE) Ser protease Ser Asp-His-Ser Ser protease acetylcholine ester bond peptide bond Ser protease subtilisin Ser
-
E6-22
Wadman (1996) Nature 383 p.753
-
E6-23
Intein & Extein: intein
(+) (protease inhibitor) (-)
ProteasomeProteasome ubiquitin ChaperoninChaperonin
Juang RH (2007) BCbasics
-
E6-24
Goldberg (1995) Science 268: 522Harvard Medical School
Proteasome ubiquitin
Lowe et al (1995) Science 268: 533
chaperonin proteasome ubiquitin proteasome
-
E6-25
Exon 1
RNARNA
Extein 1
ProteinProteinExtein 1 Extein 2InteinIntein
Exon 1 Exon 2IntronIntron
Exon 2
Extein 2Juang RH (2007) BCbasics
intron exon intein exteinintein
-
E6-26
6.2
Kinase
Phosphatase
P
OHSerSer Thr Tyr (His)
Glycogen phosphorylase b Glycogen phosphorylase a
Fischer, Kreb (1978)
Juang RH (2007) BCbasics
GP GP
GP R T GP Ser, Thr Tyr -OH His imidazole
-
-
E6-27
Insulin
Glycogen synthase
Signal TransductionSignal Transduction
Glucagon
GTP-protein-linked receptor
Tyrosine-kinase-linked receptor
Glycogen phosphorylase
Cori & Cori (1947)
Juang RH (2007) BCbasicsRef: E6-38
(glycogen synthase, GS)(glycogen phosphorylase, GP) (insulin) (glucagon)
GS GP
-
E6-28
Glycogen phosphorylase Glycogen synthaseE
nzym
e ac
tivity
0 2 4 6 8Time (min)
Adapted from Stryer (1995) Biochemistry (4e) p.597
(1) (signal)
(2)
-
E6-29
Mathews et al (1999) Biochemistry (3e) p.299
PGlycogen phosphorylase
Glycogen synthase P P
U
Glc-1-P
UDP-Glc
Glycogen
Ref: N3-3Juang RH (2007) BCbasics
-
E6-30
GP
Glycogen
n n-1
Glc-1-P Glc-6-P Glycolysis
Glycogenphosphorylase a*
Phos
phat
aseGP
kinase
Glycogenphosphorylase b
(inactive)
ATP
Proteinkinase A
cAMP
+
+
AMP (+)ATP (-)Glc-6-P (-)
Glucose (-)Caffeine (-)
P
R
T6.2
6.3
6.4
P
P
Juang RH (2007) BCbasics
Glu
cago
n
(GP) Glc-1-P
GP b (GP kinase) a (protein kinase A, PKA) GP PKA cAMP
cAMP glucagon G protein ATP cyclase ATP cAMP cAMP PKA GP kinase GP
GP GP (phosphatase) bGP ( Glc-6-P, ATP) GP
GP AMP, Glc-6-P
-
E6-31
A
A
P
P
Stryer (1995) Biochemistry (4e) p.591, 592
PA
Ser14 N-GP AMP
-
E6-32
A
AP
Ser 14
Arg 43
Arg 69
P
Stryer (1995) Biochemistry (4e) p.593
TR
GP Arg69 relaxed form (R) Arg43
tense form (T)
-
E6-33
Alberts et al (1994) Molecular Biology of the Cell (3e) p. 202, 205
Cyclin-dependent kinase (+) ATP
Cyclin (-)
Cyclin (-)
Cdk (+)
Cyclin dependent kinase (Cdk)
() Cdk ATP ()
Cdk () cyclin ATP (inhibitory P) ATP Cdk
-
E6-34
P
ATP
ADPP PGTP
GDP
GTP
GDP
Signal Out Signal Out
Signal InSignal In
ProteinKinase
ProteinPhosphatase
GEF
GAP
GTP
GDPGDP
Ada
pted
from
Alb
erts
et a
l(20
02) M
olec
ular
Bio
logy
of t
he C
ell (
4e) p
. 182
() GTP () GEF (guanine nucleotide exchange factor) GDP GTP GAP (GTPase-activating protein) GTP GDP GEF GAP
GTP Ras GTP GDP ( switch helix) tRNA ( tRNA)
GTP-GDP GTP
-
E6-35
Protein Kinase
(1) ATP (2) phosphatase(3) (4) (5)
Alberts et al (2002) Molecular Biology of the Cell (4e) p. 178, 179
5 100 250
-
E6-36
cAMP Protein Kinase A
R C
R C
R
RA
A
A
A
AA
A
A
C
C
Regulatorysubunits
Catalyticsubunits
cAMPActive kinase
C
CREB
CREB
P
ONDNA
Alb
erts
et a
l(20
02) M
olec
ular
Bio
logy
of t
he C
ell (
4e) p
. 857
, 858
protein kinase A (PKA) cAMP PKA ( CRE-binding protein, CREB)
PKA (C) (R)R cAMP
-
E6-37
G protein
cAMP (Second Messenger)
Glc-1-P
P a b
P
cAMPATP
G protein
Adenylate cyclase
A Cyclic AMP (second messenger)
Protein kinase A
Glycogen phosphorylase kinase
Glycogen phosphorylase
Glycogen
Receptor GlucagonSutherland (1971)
Cascade
Juan
g R
H (2
007)
BC
basi
cs
() cAMP glucagon glucagon receptor receptor G protein ()G protein adenylate cyclase () ATP cAMP
receptor Receptor receptor glucagon
-
E6-38
SH2domain
G protein
GDP
+ Signal
-GDP+GTP
GDP
GTP
GTP
Adenylate cyclase
+ Signal
P
ProteinPhosphatase
GlycogenSynthase
GlycogenSynthase P
active
Insulin
P P
PP kinase
Glucagon
A
G-protein-linked Receptor
Enzyme-linked ReceptorIon-channel-linked Receptor
Gilman, Rodbell (1994)
Juan
g R
H (2
007)
BC
basi
cs
receptor
(1) G-protein-linked Receptor: Receptor G protein G protein GTP GTP adenylate cyclase ATP cAMP
(2) Enzyme-linked Receptor: Receptor ( tyrosine kinase) ( SH2 domain )
(3) Ion-channel-linked Receptor:
-
E6-39
Glycogen
P
P
A
GP kinase
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
PKA
P
active
inactive
PhosphataseG
luca
gon
Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.217
receptor glucagon cAMP PKA PKA ( GP kinase, protein phosphatase inhibitor) ( glycogen synthase, protein phosphatase)
protein phosphatase () glucagon () inhibitor protein phosphatase
-
E6-40
(Ras vs. P53)
Ras
E2F
mRNA
P53
mRNA
Juang RH (2007) BCbasics
Ras E2F E2F E2F
P53 P53 () E2F
P53 P53
(Ras) (P53)
-
E6-41
6.3 cAMP Calmodulin
CalmodulincAMP
Stry
er(1
995)
Bio
chem
istry
(4e)
p.3
43
Nel
son
& C
ox(2
000)
Leh
ning
er P
rinci
ples
of B
ioch
emis
try (3
e) p
.458
O3
A
RO=P
O-
O 5P
Inositol P3
Diacylglycerol
cAMP PKA calmodulin Calmodulin
-
E6-42
Calmodulin
Alb
erts
et a
l(20
02) M
olec
ular
Bio
logy
of t
he C
ell (
4e) p
. 865
Calmodulin
CaM-kinase II calmodulin domain doamin domain domain calmodulin CaM-kinase II domain domain domain domain () domain Calmodulin
-
E6-43
Receptor
Hormone Signal
G
Cyclase
Transducer
Effector Enzyme
Effector
Effect
G-protein
Juang RH (2007) BCbasics
cyclase cAMP effector cAMPcAMP second messenger
kinase
(amplification) (flexibility)
-
E6-44
..
starch phosphorylase
-
E6-45
CCC
6.4 ATCase
+
Active relaxed form
Inactive tense form
ATCase
RR
RR
RR
CCC
COO-CH2
HN-C-COO-H H
---
-
OH2N-C-O-PO32-
= OH2N-C-
=
COO-CH2
N-C-COO-H H
---
-
Catalytic subunits
Catalytic subunits
Regulatory subunits
ATP
CTP
Feedback inhibition
AspartateCarbamoylphosphate
Carbamoyl aspartate
CTPCTP
CTPCTP
CTPCTP
Juang RH (2007) BCbasics
CTP
aspartate transcarbamoylase (ATCase) CTP CTP ATCase ( CTP ) CTP ATCase R C
CTP ATCase CTP R C ATCase relaxed form tense form
-
E6-46
ATCase
Relaxed formTense formMathews et al (2000) Biochemistry (3e) p.403
ATCase relaxed form (R) tense form (T) CTP T form ATCase (C)
-
E6-47
ATCase
Relaxed formTense form
C subunits only
Stryer (1995) Biochemistry (4e) p.241
ATCase relaxed from tense form
-
E6-48
Aspartate transcarbamoylase: (ATCase)
Allosteric enzymeAllosteric enzyme::
SS ()
CTP (-) ATP ATCase (+) ATP CTP (R) Asp SS (sigmoidal) ATCase cooperative ATCase relaxrelax (active) tensetense
Juang RH (2007) BCbasics
ATCase (CTP ATP)ATCase Michaelis-Menten S
ATP ATCase CTP CTP ATCase CTP
-
E6-49
S
Sigmoidal S
S On/Off
(ATP) S
(CTP) S
Noncooperative(Hyperbolic)
Cooperative(Sigmoidal)
CTPATP
vo
vo
[Substrate]Off On
Juang RH (2007) BCbasics
S S () ATCase
ATP ATCase S M-M CTP ATCase S
-
E6-50
T
T
R
T
[S]
vo
S S
R
R
SS
RS
A
I
T[S]
vo
[S]
vo
(+)
(-) X X
X
R = Relax(active)
T = Tense(inactive)
Allosteric siteHomotropic(+)Concerted
Heterotropic(+)Sequential
Heterotropic(-)Concerted
Allosteric site
Kinetics CooperationModels
(-)
(+)
(+)
Juang RH (2007) BCbasics
(relax) (tense) () allosteric effect
[S] homotropic () [A] (+) [A] heterotropic concerted ()sequential () [I] (-)[A] [I] allosteric site
[A] relax form S M-M [I] S
-
E6-51
PA
PA
P
P
A
A
P
P
GP kinase
GP phosphatase 1
PA
PA
P
PPA
PAA
A
A
AMP
ATPGlc-6-PGlucoseCaffeine
GlucoseCaffeine
R
T
R
T
Gar
rett
& G
risha
m (1
999)
Bio
chem
istry
(2e)
p.6
79
E6-30
Ser 14 AMP cAMPGlc-6-PATP AMP ATP AMP ATP GP GP ATP, Glc-6-P
T R R T
R T () (Glc, caffeine)
-
E6-52
PA
PA
AMP
Stryer (1995) Biochemistry (4e) p.592
AMP Ser14 N- AMP Arg242 42 45
-
E6-53
0 20 40 60 80 100 120
100
80
60
40
20
0
Myoglobin
Hemoglobin
Car
toon
by
I. G
eis
Mat
hew
s et
al (
2000
) Bio
chem
istry
(3e)
p.2
14, 2
16
(R/T) S
-
E6-54
-
E7-1
11 E1 22 E2 33 E3 44 E4 55 E5 66 E6 77 E7 88
EnzymeEnzyme
() (glycolysis)
(computational biology) (systems biology)
-
E7-2
Diabetes Mellitus (1980) Eli Lilly and Company (8e), p.20-21
-
E7-3
7.1
Alberts et al (2002) Molecular Biology of the Cell (4e) p.107 Dressler & Potter (1991) Discovering Enzymes, p.22
- (glycolysis) - (TCA cycle)
-
E7-4
(1) (2) (3) (4)
Alberts et al (2002) Molecular Biology of the Cell (4e) p.173
A
B
C
D
E
Enzyme 1Enzyme 1
Enzyme 2Enzyme 2
Enzyme 3Enzyme 3
Enzyme X Enzyme Y
Yang Cycle ()
-
-
E7-5
Citricacidcycle
7.1.2
P
P
P 2
1
$ NADH$ $ATP
H3-C-H
H3-C-OH
H2-C=O
H-COOH
O=C=O
HH4
3
2
1
0
Mitochondria
Kinase
Oxidative phosphorylation
Glycolysis
AAcetyl CoA
Pyruvate Pyruvate
STAGE 1
STAGE 2 STAGE 3
H2O
CO2
63
3
Glc-6-P GlycogenGlc-1-P Glycogen phosphorylase
OO0
1
1
2
2
Glucose
Starch
Juan
g R
H (2
007)
BC
basi
cs
(glycolysis) (pyruvate) (acetyl CoA) NADH ATP
() ()
pyruvatepyruvate acetyl CoA
(1) Glc-6-P Glycolysis ATP(2) CO2 + H2O(3) 6 3 3 1(4) (5) -OH CO2
-
E7-6
(1) (2)
NADHNADH ATPATP
CO2Stage 1 Stage 2 Stage 3
Starch Glc-6-P Acetyl CoA NADH ATP
Juang RH (2007) BCbasics
(1) (2) (3) Stages Stage (ATP) NADH NADH () ATP
-
E7-7
7.2
7.2.4 (1) (2) (3)
7.2.3 Second messenger
7.2.2 (6.1~6.4)
7.2.1 (lac operon: repressor)
Juang RH (2007) BCbasics
Metabolic pathways
Signal transduction
Cell biology
Molecular biology
second messenger ( cAMP)
-
E7-8
DNA
ribosomemRNA
proteins
7.2.1
RNA ProcessingRNA Transport
RNA Degradation
Post-translationalcontrol
proteins
cap5 3
tail
mature mRNA
DNA5
3processmRNA
Juang RH (2007) BCbasics
mRNA mRNA
-
E7-9
R
Operon
Operator Gene
S S
mRNA
RS
RNA Polymerase
Operator Gene
R
RNA Polymerase
R
PP
(S) repressor
(P) repressor
X
ON
OFF
RS
Juang RH (2007) BCbasics
operon
(S) repressor repressor
(P) P repressor operator RNA DNA regulator
-
E7-10
7.2.4
Compartmentalization
Pyruvate dehydrogenase
Mitochondria
Alb
erts
et a
l (20
02) M
olec
ular
Bio
logy
of t
he C
ell (
4e) p
.78,
99
Campbell (1999) Biochemistry (3e) p.513
tRNA
( pyruvate dehydrongenase) () (lysosome)
-
E7-11
synaptic endocrine
paracrine contact
7.2.3
Ada
pted
from
Alb
erts
et a
l (20
02) M
olec
ular
Bio
logy
of t
he C
ell (
4e) p
.833
T
receptor ()
-
E7-12
7.3
Scientific American Juang RH (2007) BCbasics
(1)
(2)
(3)
-
E7-13
Juang RH (2007) BCbasics
-
E7-14
1 2 3
9 10 11 12
6
4 85
7
4
5
8
4 6 7 85
1 3 4 6 7 8 9 10 11 122 5
Juang RH (2007) BCbasics
12 5 1, 2, 3 9, 10, 11, 12 4 ~ 8 () 7 4, 5, 8 5 4 8 5 4 8
-
E7-15
()
Cell debris
Gel filtration,SDS-PAGE,Ultrafiltration
Ion exchange,Chromatofocusing,
Disc-PAGE,Isoelectric focusing
Reverse phasechromatography,
HIC,Salting-out
Affinitychromatography,Hydroxyapatite
Juang RH (2007) BCbasics
()
(gel filtration)
-
E7-16
Proteomics
Juan
g R
H (2
007)
BC
basi
cs
Systems Biology, Integrated Biology
Proteome genome genome Genome proteome genome
-
E7-17
Juang RH (2007) BCbasics
Proteolyticdigestion
GCG
Proteolytic fragmentsPure protein2D electrophoresisSample
MALDI-TOF
CapillaryElectrophoresis
HPLC
N-Amino acid sequencing
Mass spectrum
Databasesearching
LC/MS/MS
MALDI-TOF ()
HPLC
-
E7-18
MALDI-TOF
Protease digestion
m/z
P1 P2 P3 P4
MW4 MW2 MW3 MW1
Unknown protein
Search Database
Candidate protein
Digestion Simulation
MW4 MW2 MW3 MW1
Calculate Mol wt
Juang RH (2007) BCbasics
2002
MALDI-TOF MALDI (matrix-assisted laser desorption) TOF (time of flight) TOF
(P1~P4) MALDI-TOF ( MW1~MW4) ()
-
E7-19
LC
ESI-Mass/Mass
Protease digestion
P1 P2 P3 P4
Unknown protein
P4
GK
GS
WV
R
-GKGSWVR
P1 P2 P3
ESI-Mass/Mass
P4
PLC
P4P
Protease digestion
Phosphorylated protein
Juang RH (2007) BCbasics
ESI-Mass-Mass ESI electrospray ionization MALDI pH Tandem Mass (Mass-Mass)
()
-
E7-20
4
Hem
oglo
bin
A
Hem
oglo
bin
S
TLC
TLE
Sickle cell
Linus Pauling
Pauling
Enz(1) 2007BEnz(2) 2007BEnz(3) 2007BEnz(4) 2007BEnz(5) 2007BEnz(6) 2007BEnz(7) 2007B