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The Story of Catalysis •Why Catalysis? •What is a Catalyst? •A Petrochemical Application •How Catalysts Work

Kuliah Pengantar Enzimologi Baru 1

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Page 1: Kuliah Pengantar Enzimologi Baru 1

The Story of Catalysis

•Why Catalysis?•What is a Catalyst?•A Petrochemical Application•How Catalysts Work

Page 2: Kuliah Pengantar Enzimologi Baru 1

Why Catalysis?

Need to make chemicals faster

Most Reactions are too slow to be useful...

Page 3: Kuliah Pengantar Enzimologi Baru 1

What is a Catalyst?

Catalysts speed up a chemical reaction without being used up...

Catalyst + Reactants

Catalyst-Reactants

Catalyst + Products

Page 4: Kuliah Pengantar Enzimologi Baru 1

The Nature of Enzyme Catalysis

●● Enzyme provides a catalytic surfaceEnzyme provides a catalytic surface

●● This surface stabilizes transition stateThis surface stabilizes transition state

●● Transformed transition state to productTransformed transition state to product

B

BA Catalytic surface

A

Juang RH (2004) BCbasics

Page 5: Kuliah Pengantar Enzimologi Baru 1

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 state

Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252

X

Page 6: Kuliah Pengantar Enzimologi Baru 1

Examples of Reactions w/o Catalysis

Oxygen in theair mixes with iron to formrust

HydrogenPeroxideforms a chemicalreaction withyour body

1 week to several months

Plants & animals decompose to oil & coal

Millions of years

Seconds

Page 7: Kuliah Pengantar Enzimologi Baru 1

Ways to Make Ways to Make Chemicals FasterChemicals Faster

Add other Chemicals Catalysts!!!!

Disadvantage--Too hot!

Disadvantage--Separate chemicals Disadvantage--Costly

Pressure

Disadvantage--Cause Explosions

Temperature

Page 8: Kuliah Pengantar Enzimologi Baru 1

Enzyme Stabilizes Transition State

S

P

ES

EST

EP

ST

Reaction direction

Energy change

Energy required (no

catalysis)

Energy decreases (under

catalysis)

What’s the difference?T = Transition state

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166

Page 9: Kuliah Pengantar Enzimologi Baru 1

Active Site Is a Deep Buried Pocket

Why energy required to reach transition stateis lower in the active site?

It is a magic pocket

(1) Stabilizes transition(2) Expels water(3) Reactive groups(4) Coenzyme helps

(2)

(3)(4)

(1)CoE

+

-

Juang RH (2004) BCbasics

Page 10: Kuliah Pengantar Enzimologi Baru 1

Enzyme Active Site Is Deeper than Ab Binding

Instead, active site on enzymealso recognizes substrate, butactually complementally fits the transition state and stabilized it.

Ag binding site on Ab binds to Agcomplementally, no further reactionoccurs.

Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252

X

Page 11: Kuliah Pengantar Enzimologi Baru 1

Active Site Avoids the Influence of Water

Preventing the influence of water sustains the formation of stable ionic bonds

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115

-+

Page 12: Kuliah Pengantar Enzimologi Baru 1

Reaction RatesReaction Rates

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14

Time

En

erg

y

Without Catalyst

With Catalyst

Page 13: Kuliah Pengantar Enzimologi Baru 1

Cyclar...An Example of A Catalytic

Process

2 ( C - C - C ) + Catalyst + Catalyst

Propane Benzene

• Propane is unreactive• Benzene is in higher demand than propane

Page 14: Kuliah Pengantar Enzimologi Baru 1

How Does Cyclar Work?

Surface Area!!

Catalysts are not solid Catalysts are porous

Page 15: Kuliah Pengantar Enzimologi Baru 1

What Does Surface Area Do?

Increases the amount of available reaction sites

2 propanes

Reactive Sites

Benzene Benzene product

Page 16: Kuliah Pengantar Enzimologi Baru 1

Do Catalysts Live Forever?

NO!!!Catalysts can last from 2 hours to 2 years

They can die from:--poisons which contaminate the catalyst--large molecules which cover the catalyst --over heating, over pressurizing--crumbling/crushed

Page 17: Kuliah Pengantar Enzimologi Baru 1

Conclusions

Catalysts are reusable Catalysts help increase reaction rates

Catalysts work using surface area

Page 18: Kuliah Pengantar Enzimologi Baru 1

Who Uses Catalyst?Who Uses Catalyst?

Oil Industries

Your body (Enzymes)

Chemical/Pharmaceutical Companies

Catalytic Converters in your car

Page 19: Kuliah Pengantar Enzimologi Baru 1

Examples of Reactions w/Catalysts

C - C - C +

Propane + Catalyst = Benzene + Catalyst

= +

+ =ProteinsFatsSimple Sugars

+

Food + Enzymes = Small Molecules + Enzymes

Page 20: Kuliah Pengantar Enzimologi Baru 1

Examples of Catalysis

Basic substances like ashes catalyze the burning of sugar

A non-reactive substance like water turns reactive when mixed with catalyst

Page 21: Kuliah Pengantar Enzimologi Baru 1

What Makes a Catalyst Work?

Surface Area!!!!

Steel Wool has a large surface area, so it oxidizes easily

Page 22: Kuliah Pengantar Enzimologi Baru 1

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Ca

rtoo

n G

uid

eEq

uatio

n an

d De

scrip

tion

[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].

[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.

[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].

E + S → ES → E + P + II↓EII

E + S → ES → E + P + + II II↓ ↓EII + S →EIIS

↑ ↑

E + S → ES → E + P + II ↓ EIIS

EI

S X

Juang RH (2004) BCbasics

Page 23: Kuliah Pengantar Enzimologi Baru 1

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Km’ [S], mM

vo

[S], mM

vo

II II

Km [S], mM

Vmax

II

Km’

Vmax’Vmax’

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax & Km decreased

II

1/[S]1/Km

1/vo

1/ Vmax

II

Two parallellines

II

Intersect at X axis

1/vo

1/ Vmax

1/[S]1/Km 1/[S]1/Km

1/ Vmax

1/vo

Intersect at Y axis

= Km’

Juang RH (2004) BCbasics

Page 24: Kuliah Pengantar Enzimologi Baru 1

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-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-

Page 25: Kuliah Pengantar Enzimologi Baru 1

Sulfa Drug Is Competitive Inhibitor

-COOHH2N-

-SONH2H2N-

PrecursorFolicacid

Tetrahydro-folic acid

SulfanilamideSulfa 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)

Page 26: Kuliah Pengantar Enzimologi Baru 1

Enzyme Inhibitors Are Extensively Used

●● Sulfa drug (anti-inflammation)

Pseudo substrate Pseudo 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 Symmetry

Notsymmetry

→ Human aspartyl protease:(monodimer)

domain 1

Asp Asp

domain 2

subunit 2

Asp

subunit 1

Asp

Juang RH (2004) BCbasics

Alzheimer's disease

Page 27: Kuliah Pengantar Enzimologi Baru 1

HIV protease vs Aspartyl protease

Asymmetric monomer

↓ HIV protease HIV protease (homodimer)

HIV Protease inhibitor is used in treating AIDS

Symmetricdimer

Asp

subunit 2

↑Aspartyl protease (monomer)

subunit 1

Asp

domain 1 domain 2

Asp Asp

Juang RH (2004) BCbasics

Page 28: Kuliah Pengantar Enzimologi Baru 1

O ON–C–C–N–C–C N–C–C–N–C–C R H R’

Chymotrypsin Has A Site for Specificity

O -

CSer

Active SiteActive Site

SpecificitySite

SpecificitySite Catalytic Site

Juang RH (2004) BCbasics

Page 29: Kuliah Pengantar Enzimologi Baru 1

Specificity of Ser-Protease Family

COO-

CAsp

COO-

CAsp

Active Site

Trypsin Chymotrypsin Elastasecut at Lys, Arg cut at Trp, Phe, Tyr cut at Ala, Gly

Non-polarpocket

De

ep

and

neg

ativ

ely

cha

rged

poc

ket

Shallow andnon-polar

pocket

O O–C–N–C–C–N– C C C C NH3+

O O–C–N–C–C–N– C

O O–C–N–C–C–N–

CH3

Jua

ng

RH

(2

00

4)

BC

ba

sics

Page 30: Kuliah Pengantar Enzimologi Baru 1

Stereo Specificity

BC

DBC

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

Page 31: Kuliah Pengantar Enzimologi Baru 1

Control Points of Gene Regulation

Prokaryotics

DNA

ribosomemRNA

proteins

Post-translationalcontrol Eukaryotics

proteins

cap5’ 3’

tail

mature mRNA

DNA

5’3’process

mRNA

Juang RH (2004) BCbasics

Translation

Activity

Proteolysis

Transcription

RNA ProcessingRNA Transport

RNA Degradation

Page 32: Kuliah Pengantar Enzimologi Baru 1

xRegulatory

subunit

o

Regulation of Enzyme Activity

o xS I

x oS

Sx

S

oS

AA

Po R xR

+

III

or

inhibitor

proteolysis

phosphorylation

cAMP orcalmodulin

or

regulatoreffector

P

(-)

(+)

Inhibitor Proteolysis

Phosophorylation

Signal transduction

Feedback regulation

Jua

ng

RH

(2

00

4)

BC

ba

sics

Page 33: Kuliah Pengantar Enzimologi Baru 1

Cascade Amplification of Signals

Cascade

nS nP1 Enzyme

Juang RH (2004) BCbasics

Page 34: Kuliah Pengantar Enzimologi Baru 1

How

to S

epara

te T

hese

Ob

jects

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 cotton

cotton

wood

stone

ShapeSizeDensity

Shape

Density

Size

Sieving different sizes Different sedimentationDifferent rolling speed

4 6 7 85

1 3 4 6 7 8 9 10 11 122 5

Juang RH (2004) BCbasics

Page 35: Kuliah Pengantar Enzimologi Baru 1

Basic Principles of Protein Purification

Ammonium sulfate fractionation

Cell OrganelleHomogenization

MacromoleculeNucleic

acid Carbohydrate (Lipid)

Size Charge Polarity Affinity

Small molecule Cell DebrisProtein

Amino acid, Sugar,

Nucleotides, etc

Gel filtration,SDS-PAGE,Ultrafiltration

Ion exchange,Chromatofocusing,

Disc-PAGE,Isoelectric focusing

Reverse phasechromatography,

HIC,Salting-out

Affinitychromatography,Hydroxyapatite

Juang RH (2004) BCbasics