enzyme_revised 2011 chatchawin

8/6/2019 Enzyme_revised 2011 Chatchawin

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EnzymeEnzyme

Chatchawin Petchlert, Ph.D.

Department of Biochemistry

Faculty of Science, Burapha University

ChymotrypsinChymotrypsin


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OUTLINEOUTLINE

xGENERAL PROPERTIES

xCOFACTORS

xCHEMISTRY OF CATALYST

xENZYME KINETICS

xENZYME INHIBITION

xENZYME REGULATIONS


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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.


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4

EnzymeEnzyme

Enzyme-substrate complex

Ribozyme

Abzyme


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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


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6


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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.


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8

10-5 M

10-4 10-3 M

Turnover number (S)

(P)


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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)


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10

(High specificity)

2 - (substrate

specificity)- (reaction

specificity)

- (chemical catalyst)


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veravera

reactionreactionE (enzyme) +S (substrate) Step I :

Binding

ES(enzyme-substratecomplex)

Step II :Transformation

E +P roduct


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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


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13

6 oxidoreductase dehydrogenase, transferase, hydrolase, lyase,isomerase ligase 3 (trivial name) (systematicname)

1 : 2 ase lactatepyruvate NAD+ 2


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14


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15

6

1 Oxidoreductase (oxidation-reduction) (redox reaction)

Aox + Bred Ared + Box

2 Transferase


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16

()

3 Hydrolase (hydrolysis)

AB + H2O AOH + BH

4 Lyase

ABC AB + C


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17

()

5 Isomerase

ABC BAC

6 Ligase 2

ATP


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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


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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)


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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


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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)


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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


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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


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Induced fit modelInduced fit model

E i ti 2 t iti


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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


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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


E St bili T iti St t


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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


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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

+

-


Stickase


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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


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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


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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

-+


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Enzyme KineticsEnzyme Kinetics

Enzyme Kinetics


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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



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36

Invertase (IT)


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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


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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


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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


Constant ES Concentration at Steady State


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Constant ES Concentration at Steady State

S P

EES

Reaction Time

Con

centra

tion


An Example for Enzyme Kinetics (Invertase)


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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


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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


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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


Significance of Enzyme Kinetics


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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


K : Affinity with Substrate


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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


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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



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Turn Over Numbers of Enzymes


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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


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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


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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)


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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


Enzyme Inhibition (Plots)


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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


Competitive Inhibition


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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


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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


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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


Alzheimer's disease

HIV protease vs Aspartyl protease


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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



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Enzyme CatalysisEnzyme Catalysis

Chymotrypsin Catalytic Mechanism A1


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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


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Chymotrypsin Catalytic Mechanism A3


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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


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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


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H

y yp y

O

O

C

C

N CC

[NH2]CC

H

Second Transition State

OH


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Chymotrypsin Is Activated by Proteolysis


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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


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pH Influences Chymotrypsin Activity


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5 6 7 8 9 10 11

pH

RelativeAc

tivity

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162


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Chymotrypsin Ser195 Inhibited by DIFP


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O

(CH3)2CHOPOCH(CH3)2

F

=

Diisopropyl-fluorophosphate (DIFP)


O-H

CH2

Ser 195

O

(CH3)2CHOPOCH(CH3)2

=

O

CH2

Ser 195

XXXX


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Chymotrypsin Also Catalyzes Acetate


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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


Two-Stage Catalysis of Chymotrypsin


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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)



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Active Site Stabilizes Transition State


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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


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Basic Mechanism of Catalysis


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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


Concerted Mechanism of Catalysis


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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


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O ONCCNCC NCCNCC

R H R

O-

C

Ser

Active SiteActive Site

Specificity

Site

Specificity

Site Catalytic Site


Specificity of Ser-Protease Family


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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

pocket

O O

CNCCN

C

CC

C

NH3

+

O O

CNCCN

C

O O

CNCCN

CH3

Jua

ngRH

( 2004)BCbasics

Control Points of Gene Regulation


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Prokaryotics

DNA

ribosomemRNA

proteins

Post-translationalcontrol

Eukaryotics

proteins

cap5 3

tail

maturemRNA

DNA

53process

mRNA


Translation

Activity

Proteolysis

TranscriptionRNA Processing

RNA Transport

RNA Degradation

Regulation of Enzyme Activity


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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


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Cascade

nS nP1 Enzyme



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85


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86


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88


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90


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91

Two views of the regulatory enzyme aspartate trans-carbamoylase (derived from PDB ID 2AT2) The regulatory


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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.


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Phosphorylation

Fischer Kreb (1978)


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Conformational

ChangedephosphorylastionPhosphatase

P

Protein

OH

SerSerThr Tyr(His)

Active Inactive

Inactive Active

Glycogen phosphorylase b Glycogen phosphorylase a

Fischer, Kreb (1978)


Kinasephosphorylation

Regulation of Blood Sugar

Cori & Cori (1947)


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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)



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Glycogen Phosphorylase, GP


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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



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cAMP Is the Second Messenger

GlSutherland (1971)


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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)


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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


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


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Receptor

Hormone Signal

G

Cyclase

Transducer

Effector Enzyme

Effector

Effect

G-protein


Allosteric Enzyme ATCase

Active relaxed form


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


Quaternary structure

Sigm

Positive effector

Noncooperative

(Hyperbolic)

vo


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


Mechanism and Example of Allosteric Effect


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

(-)

(+)

(+)


Activity Regulation of Glycogen Phosphorylase


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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


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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


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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


Cross Talk between Cells

Direct contact Diffusion


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(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


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


Basic Principles of Protein Purification


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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,

enzyme_revised 2011 chatchawin

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