aulani " biokimia enzim " presentasi 1 1 basic enzyme aulanni’am biochemistry laboratory...

Aulani " Biokimia Enzim " Presentasi 1 1

Basic enzyme

Aulanni’am

Biochemistry Laboratory

Brawijaya University


What are enzymes ?

Enzymes are proteins They have at least one active site Active site is lined with residues and sometimes

contains a co-factor Active site residues have several important properties:

Charge [partial, dipoles, helix dipole] pKa Hydrophobicity Flexibility Reactivity (Cysteines)


What are chemical reactions?

In a chemical reactions a compound “A” is changed into a compound “B”.

In context of biochemistry, chemical reactions are “organic chemistry reactions”.

In organic chemistry reactions bonds are broken and/or formed (generalization)

Bonds are “paired electrons” between two nuclei (C-C, C=C, C-O,C=O, C-H, O-H, N-H etc.)

Thus reactions involve “rearranging” electrons In context of biochemistry, a frequent player in

chemical reactions is H2O (hydronium H3O+ and hydroxide OH-)


Enzyme catalysis

Enzyme catalysis is characterized by two features

Substrate specificity Rate acceleration


Enzyme substrate specificity

Unlike “chemical catalysts” enzyme only catalyze reactions for a “relatively” narrow substrate spectrum.For example: substrate spectrum of restriction enzymes, and protein kinases.

Two main theories for substrate specificity Lock-and-Key hypothesis (Fisher, 1894) Induced-fit hypothesis (Koshland, 1958)


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

X


The Nature of Enzyme Catalysis

●● Enzyme provides a catalytic surface

●● This surface stabilizes transition state

●● Transformed transition state to product

B

BA Catalytic surface

A


Lock-and-Key vs. Induced-Fit

Lock-and-Key does not always explain substrate spectrum (e.g. analogs smaller than substrate don’t bind while analogs larger than substrate do bind)

Induced-fit implies the concepts: conformational change catalytically competent conformation (low

catalytic form and high catalytic form)


Catalyzed vs. un-catalyzed reactions

Reaction CoordinateReaction Coordinate

Fre

e E

nerg

y (d

elta

G)

S

P

S‡

S‡c

ES‡

ES

EP


HO

H

Induced to transition state

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 Acid

catalysis

Basecatalysis

Both

NH

+

C- OO

HO

H

Specific


Rate Acceleration

Catalyzes of a reaction results in rate enhancement not alteration of the equilibrium

Catalysis involves reduction of activation energy

This can be most readily done by lowering the Free Energy of the transition state

Additionally the Free Energy of the ground state can be raised (not a general strategy)

S‡

ES‡

ES

EP

Reaction Coordinate

Fre

e E

nerg

y (d

elta

G)

S

P


Transition state Stabilization by Enzyme

How does an Enzyme reduce the Activation Energy ??

Enzyme stabilizes the transition state, i.e. makes the “strained” conformation more bearable.

Note: An enzyme can only reduce the

activation energy if it binds better to the transition state than to the substrate

[otherwise, the DDG between ES and ES‡ is the same as between S and S‡]

S‡

ES‡

ES

EP

Reaction CoordinateF

ree

Ene

rgy

(del

ta G

)

S

P


Transition state Stabilization by Enzyme

Implications of preferential stabilization of the transition state.

Compounds that closely mimic the transition state bind much better to an enzyme than the original substrate.

Transition state analogs are potent inhibitors (pico molar affinities)

S‡

ES‡

ES

EP

Reaction Coordinate

Fre

e E

nerg

y (d

elta

G)

S

P

Applications:

• Inhibitor/drug development based on transition state model

• Development of catalytic antibodies [rate acceleration up to 105]


Enzyme Stabilizes Transition State

S

P

ES

EST

EP

ST

Reaction direction

Energy change

Energ

y re

quire

d (n

o

cata

lysis)

Energ

y d

ecre

ase

s (under

cata

lysis)

What’s the difference?T = Transition state


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

+

-


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.

X


Enzyme mediated catalysis

Strategies for transition state stabilization and/or ground state destabilization: Proximity Strain or distortion Orbital steering

However, additionally the enzyme can be an “active” participant in reaction Acid/base catalysis Nucleophilic/electrophilic catalysis Covalent catalysis


Rate Acceleration: Proximity

For un-catalyzed reactions involving two substrates the rate can be increased by increasing the number of collisions (higher temperature)

Enzymes capture each substrate (sometimes in a ordered manner) and appropriately orient them with respect to each other, thus obviating the need for higher temperature

The capture of substrates by the enzyme has an Entropic cost; this cost must be compensated by favourable interactions between enzyme and substrates

The effect of confining the substrates in the active site of the enzyme is similar to raising the concentration of the substrates. Hence, the proximity effect is also referred to as increasing the effective concentration


Active Site Avoids the Influence of Water

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

-+


Essential of Enzyme Kinetics

E S+ P+

Steady 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

S P

EES

Reaction Time

Con

cen

tratio

n


The “Active” EnzymeExamine the hydrolysis of an ester:

O

ORR'

+ H2OR

O

OH

HO R'+very slow

Weak electrophile Poor nucleophile

Expected transition state

O

ORR'

O

H H

O

ORR'

O

H H


The “Active” EnzymeBase catalyzed hydrolysis of an ester:

O

ORR'

O-

H

O

OHR

O

O-R

HO R'+ +-O R'

Catalysis is accelerated by altering the poor nucleophile H2O into a strong nucleophile OH-


The “Active” EnzymeAcid catalyzed hydrolysis of an ester:

Catalysis is accelerated by altering the weak electrophile C into a strong nucleophile C+

O

ORR'

+ H3O+

+OH

ORR' C+

OH

ORR'

C

OH

ORR'

O

H H

O

OHR

HO R'+


The “Active” Enzyme

In standard organic chemistry for ester hydrolysis one has to choose between base or acid catalysis

In enzyme catalysis the reaction is “carried out on a solid support”

As a consequence one can incorporate both acid and base catalysis:


The “Active” EnzymeEnzyme catalyzed hydrolysis of an ester:

Active site incorporates both:

• a base [-B:]

• an acid [-B+-H]

O

ORR'

O

H HB:

B+

H

-O

HB+

B:

H

C

OH

ORR'

C

O

R

O

HB+

B:

H

HO R'

O

ORR'

O

H HB:

B+

H


Catalysis of Phosphorylation

Phosphorylation a very frequent reaction (e.g. signal transduction)

Phosphoryl donating group is generally a nucleotide, e.g. ATP, GTP

Transfer of phosphoryl group to: Water : hydrolysis [ATPase, GTPase] Anything else: phosphorylation [Kinase]


Mechanisms of Enzyme Catalyzed Phosphorylation

Several mechanism are observed in Nature Reactions with covalent enzyme intermediates Direct inline transfer Perhaps metal assisted mechanisms

Present two examples: Aminoglycoside kinases (Cousin of Protein

kinases) G-proteins


An Example for Enzyme Kinetics (Invertase)

Vmax

Km S

vo

1/S

1vo

Double reciprocal Direct plot

1)1) Use predefined amount of Enzyme → E

2)2) Add substrate in various concentrations → S (x

3)3) Measure Product in fixed Time (P/t) → vo (y

4)4) (x, y) plot get hyperbolic curve, estimate → Vmax

5)5) When y = 1/2 Vmax calculate x ([S]) → Km

1Vmax

- 1 Km

1/2


A Real Example for Enzyme KineticsD

ata

no

1234

0.250.501.02.0

0.420.720.800.92

Absorbance v (mole/min)[S]

0.210.360.400.46

(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/v421

0.5

2.081.561.351.16

→→ → →

1.0

0.5

0

v

Dire

ct p

lot

Dou

ble

reci

proc

al 2.0

1.0

0

1/v

-4 -2 0 2 41/[S]

0 1 2[S]

1.0

-3.8


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


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’


Ser195

His 57

Asp 102

H–O–CH2

O

C–O -

=

Active Ser

H–N N

C C

C

H

H

CH2

Ser195

His 57

Asp 102

- O–CH2

OC–O–H

=

N N–H

C C

C

H

H

CH2


pH Influences Chymotrypsin Activity

5 6 7 8 9 10 11

pH

Relative

Activity


pH

Influ

en

ces N

et C

harg

e o

f Pro

tein

+Net Charge of a Protein

Buffer pH

Isoelectric point,pI

-

3

4

5

6

7

8

9

10

0+


Imidazole on Histidine Is Affected by pH

H–N N

C C

C

H

H

H+

pH 6 pH 7

+H–N N–H

C C

C

H

H

Inactive+ Ser

195

His 57

Asp 102

H–O–CH2

O

C–O -

=

H–N N–H

C C-H

C

CH2

H


Chymotrypsin Produces New Ile16 N-Terminal

I16L13 Y146

Asp 194

–CH2COO-

Ile 16NH2–

Ile 16+ NH3–

5 6 7 8 9 10 11pH

Relative activity

pH 9 pH 10pKa

New NH2-terminus


New Ile16 N-Terminal Stabilizes Asp194

Asp 102

His 57 Ser 195

Asp 194

Gly 193

Ile 16

+NH3

Catalytic Triad


O (CH3)2CH–O– P –O–CH(CH3)2

F

=

Chymotrypsin Ser195 Inhibited by DIFP

Diisopropyl-fluorophosphate (DIFP)

O-…H

CH2

Ser 195

O (CH3)2CH–O– P –O–CH(CH3)2

=

O

CH2

Ser 195

XXXX


Addition of Substrate Blocks DIFP Inhibition

Reaction time

Percent Inhibition of activity (%

)

100

50

0

No substrate

Add substrate

S

+ DIFP

+ DIFP & substrate

XXXX


Chymotrypsin Also Catalyzes Acetate

O-C N- H

O-C O-

Peptide bond

Ester bond

O

CH3–C–O– –NO2

Nitrophenol acetate

HO– –NO2

O

CH3–C–OH

Hartley & Kilby

Chymotrypsin+ H2O

Nitrophenol

Acetate

No acetate was detected at early stage


O -

C

Time (sec)N

itrop

hen

ol

Two-Stage Catalysis of Chymotrypsin

O

CH3–C–O– –NO2

Nitrophenol acetate

OC

O

CH3–C HO– –NO2

+ H2O

O-HC

CH3COOH

Kinetics of reaction

Two-phasereaction

Acylation

Deacylation (slow step)


Extra Negative Charge Was Neutralized

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


Active Site Stabilizes Transition State

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


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


Classification of Proteases

MetalProtease

SerineProtease

CysteineProtease

AspartylProtease

Carboxy-peptidase A

ChymotrypsinTrypsin

Papain

PepsinRenin

H57H57

D102D102

S195-OS195-O--

C25-SC25-S--

H195H195

D215D215

D32D32H2O

Non-specific

Non-specific

AromaticBasic

Non-polar

EDTAEGTA

DFPTLCKTPCK

PCMBLeupeptin

Pepstatin

Family Example Mechanism Specificity Inhibitor

E72E72 H69H69

Zn2+

H196H196


Serine Protease and AchEChymotrypsin – 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 – – – – – – – –

Ser

19

5

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


Sig

moid

al C

urv

e E

ffect

Sigmoidal curve

Exaggeration of sigmoidal curveyields a drastic zigzag line that shows the On/Off point clearly

Positive effector (ATP)brings sigmoidal curveback to hyperbolic

Negative effector (CTP)keeps

Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity

Noncooperative(Hyperbolic)

Cooperative(Sigmoidal)

CTPATP

vo

vo

[Substrate]Off On


T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

IT

[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic

(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics CooperationModels

(-)

(+)

(+)


Activity Regulation of Glycogen Phosphorylase

PA

PA

P

P

A

A

Covalent modificationCovalent modification

P

P

GP kinase

GP phosphatase 1

No

n-co

valent

No

n-co

valent

PA

PA

P

P

PA

PAA

A

A

AMP

ATPGlc-6-PGlucoseCaffeine

GlucoseCaffeine

spontaneously

R

T

R

T

aulani " biokimia enzim " presentasi 1 1 basic enzyme aulanni’am biochemistry laboratory...

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