Enz 2007B.pdf

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+


E1-7

1

FeCl3

Hemoglobin

Catalase

1,000,000,000

1,000,000

1,000

2H2O2 2H2O + O2

P

ES


( 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 ( )


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


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


() () () () RNA

-in zyme (IUBMB) (Enzyme Commission, EC)

E2-1

2.1

Apo-enzymeApo-

enzyme Cofactor

Coenzyme

Holoenzyme


(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

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)


(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 ()


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-

-

+-

+-


( 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


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

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


()

(abzyme)

E3-4

S

P

ES

EST

EP

ST

T = Transition state


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


sp3sp2

(T)

E3-8

AbzymeAbzyme

peptide bond

: sp2 [sp3]* sp2

Why ?Why ?Abzyme

sp3

sp3


peptide bond -C-N- sp2 peptide bond sp2 C-OH sp3 sp3

albumin

Nature 383: 23 (1996)

- -

E3-9


() sp3

E3-10


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)

+

-


(1)

(2)

(3) ( Ser-OH) ( His H+)( Ser-O-)

pH ( E5-2)

(4)

E3-13

Hexokinase


Hexokinase

domain domains

E3-14


-+

()

E3-15

3

0 1 2 3 4 0 1 2 3 4


() () (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

(

)


()

E3-19

E S+ P+

Steady State TheorySteady State Theory

ES [ES]

SE E


Michaelis Menten [E] [S] [ES] [P][ES] [ES] Steady state theory [ES]

E3-20

Steady State ES

S P

EES


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


[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


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


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


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


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


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)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)


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)


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


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


E4-3

4

Competitive

Non-competitive

Uncompetitive

Penicillin

(Hg, Pb)

DFP, TPCK Sarin (-Ser) PCMB (-Cys)

(Mixed inhibition)


() ()

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


(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


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


(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


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


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


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)


cysteine protease Cysserine protease Ser ( Cys, Ser) cysteine protease PCMB Cys

E4-18

GlycopeptideTranspeptidase

(GT)


tetrapeptide

pentaglycine

Sugar chain

(glycopeptide transpeptidase, GT) GT GT

E4-19

Penicillin GT


OHSer

Glycopeptidetranspeptidase

(inactive)

OH

HC C C

O=C

HN C=OR

N CH

S CH3CH3COO-

H

O Ser

Glycopeptidetranspeptidase


(GT) Ser Ser -OH GT Ser

E4-20

Penicillin



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-


() ()

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


Arg 145

Tyr 248

Glu 270

Carboxypeptidase A Tyr248

E5-6


- -

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)


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


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


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


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


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)


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


XXX

DIFP chymotrypsin

E5-18

Asp102

His57

Ser195


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


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


-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)


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


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


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)


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


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


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


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)


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


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


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


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


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


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


AchE

O-

CH

OCH3C

CH3OCH2CH2NCH3H CH3

+

Deacylation

Acylation

Acetylcholinesterase Ser His Asp acylation deacylation

E6-20

O-

C

Sesame Triad

Hi, Everybody!Hi, Everybody!


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


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


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)


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


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)


(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


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


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

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


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


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


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


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


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


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


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

(-)

(+)

(+)


(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


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

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)


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


(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)


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


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


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


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


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


()

(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


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


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


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

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