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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:

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Basic enzyme

Basic enzyme

Aulanni’am

Biochemistry Laboratory

Brawijaya University

Aulani " Biokimia Enzim " Presentasi 1


What are enzymes

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)

Aulani " Biokimia Enzim " Presentasi 1


What are chemical reactions

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 reactionsbonds 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-)

Aulani " Biokimia Enzim " Presentasi 1


Enzyme catalysis

Enzyme catalysis

Enzyme catalysis is characterized by two features

  • Substrate specificity

  • Rate acceleration

Aulani " Biokimia Enzim " Presentasi 1


Enzyme substrate specificity

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)

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Substrate

Transition state

Product

If enzyme just binds substrate

then there will be no further reaction

X

Enzyme not only recognizes substrate,

but also induces the formation of transition state

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

B

A

The Nature of Enzyme Catalysis

●Enzyme provides a catalytic surface

●This surface stabilizes transition state

●Transformed transition state to product

B

A

Catalytic surface

Aulani " Biokimia Enzim " Presentasi 1


Lock and key vs induced fit

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)

Aulani " Biokimia Enzim " Presentasi 1


Catalyzed vs un catalyzed reactions

S‡

S‡c

Free Energy (delta G)

ES‡

S

ES

EP

P

Reaction Coordinate

Catalyzed vs. un-catalyzed reactions

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

+

+

N

H

N

H

O

=

O

O

C

=

=

O

C

C

H

C

H

=

N

H

H

C

H

H

C

H

C

N

H

N

H

H

C

H

O

O

O

N

H

O

H

H

H

H

H

H

H

H

-

-

O

O

O

O

C

C

Induced to transition state

Specific

Acid-base

Catalysis

Acid

catalysis

+d

Both

-d

Base

catalysis

Slow

Fast

Fast

Very Fast

Aulani " Biokimia Enzim " Presentasi 1


Rate acceleration

S‡

Free Energy (delta G)

ES‡

S

ES

EP

P

Reaction Coordinate

Rate Acceleration

  • Catalyzes of a reaction results in rate enhancementnot 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)

Aulani " Biokimia Enzim " Presentasi 1


Transition state stabilization by enzyme

S‡

Free Energy (delta G)

ES‡

S

ES

EP

P

Reaction Coordinate

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‡]

Aulani " Biokimia Enzim " Presentasi 1


Transition state stabilization by enzyme1

S‡

Free Energy (delta G)

ES‡

S

ES

EP

P

Reaction Coordinate

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)

  • Applications:

  • Inhibitor/drug development based on transition state model

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

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Reaction direction

Enzyme Stabilizes Transition State

Energy change

ST

Energy decreases (under catalysis)

Energy required (no catalysis)

EST

S

ES

P

EP

T = Transition state

What’s the difference?

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

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

CoE

(2)

(1)

(3) Reactive groups

(4)

(4) Coenzyme helps

-

(3)

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Enzyme Active Site Is Deeper than Ab Binding

Instead, active site on enzyme

also recognizes substrate, but

actually complementally fits the transition state and stabilized it.

Ag binding site on Ab binds to Ag

complementally, no further reaction

occurs.

X

Aulani " Biokimia Enzim " Presentasi 1


Enzyme mediated catalysis

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

Aulani " Biokimia Enzim " Presentasi 1


Rate acceleration proximity

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

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Active Site Avoids the Influence of Water

+

-

Preventing the influence of water sustains the formation of stable ionicbonds

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

E

S

Essential of Enzyme Kinetics

Steady State Theory

E

E

+

+

P

S

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.

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Concentration

Reaction Time

Constant ES Concentration at Steady State

S

P

ES

E

Aulani " Biokimia Enzim " Presentasi 1


The active enzyme

Weak electrophile

Poor nucleophile

The “Active” Enzyme

Examine the hydrolysis of an ester:

Expected transition state

Aulani " Biokimia Enzim " Presentasi 1


The active enzyme1

The “Active” Enzyme

Base catalyzed hydrolysis of an ester:

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

Aulani " Biokimia Enzim " Presentasi 1


The active enzyme2

The “Active” Enzyme

Acid catalyzed hydrolysis of an ester:

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

Aulani " Biokimia Enzim " Presentasi 1


The active enzyme3

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:

Aulani " Biokimia Enzim " Presentasi 1


The active enzyme4

The “Active” Enzyme

  • Enzyme catalyzed hydrolysis of an ester:

  • Active site incorporates both:

  • a base [-B:]

  • an acid [-B+-H]

Aulani " Biokimia Enzim " Presentasi 1


Catalysis of phosphorylation

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]

Aulani " Biokimia Enzim " Presentasi 1


Mechanisms of enzyme catalyzed phosphorylation

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

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

1

vo

vo

- 1

Km

1

Vmax

1/S

S

An Example for Enzyme Kinetics (Invertase)

1) Use predefined amount of Enzyme→ E

2)Add substrate in various concentrations→ S (x

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

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

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

Vmax

1/2

Km

Double reciprocal

Direct plot

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

A Real Example for Enzyme Kinetics

Substrate

Product

Velocity

Double reciprocal

2.0

1.0

0

1.0

0.5

0

v

1/v

1.0

-3.8

-4 -2 0 2 4

0 1 2

[S]

1/[S]

Data

v (mmole/min)

[S]

Absorbance

1/S

1/v

no

0.25

0.50

1.0

2.0

0.21

0.36

0.40

0.46

0.42

0.72

0.80

0.92

4

2

1

0.5

2.08

1.56

1.35

1.16

1

2

3

4

(1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole

(2) Reaction time was 10 min

Direct plot

Double reciprocal

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Enzyme Inhibition (Mechanism)

E + S→ES→E + P

+

I

EI

E + S→ES→E + P

+ +

II

↓ ↓

EI+S→EIS

E + S→ES→E + P

+

I

EIS

Competitive

Uncompetitive

Non-competitive

E

Substrate

E

X

Cartoon Guide

Compete for

active site

Inhibitor

Different site

Equation and Description

[I] binds to free [E] only,

and competes with [S];

increasing [S] overcomes

Inhibition by [I].

[I] binds to [ES] complex

only, increasing [S] favors

the inhibition by [I].

[I] binds to free [E] or [ES]

complex; Increasing [S] can

not overcome [I] inhibition.

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Enzyme Inhibition (Plots)

Uncompetitive

Competitive

Non-competitive

Vmax

Vmax

vo

Vmax’

Vmax’

I

Direct Plots

Km

[S], mM

Km’

Km

[S], mM

1/vo

1/vo

1/vo

I

I

Double Reciprocal

Two parallel

lines

Intersect

at X axis

Intersect

at Y axis

1/Vmax

1/Vmax

1/Vmax

1/Km

1/[S]

1/Km

1/[S]

1/Km

1/[S]

Vmax

vo

I

I

Km

Km’

[S], mM

= Km’

Vmax unchanged

Km increased

Vmax decreased

Km unchanged

Both Vmax & Km decreased

I

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

H

O

C–O–H

O

C–O-

C

=

=

N

H–N

H–O–CH2

C

C

H

CH2

H

C

Ser

195

N–H

N

-O–CH2

C

C

Asp 102

H

CH2

His 57

Ser

195

Asp 102

His 57

Active Ser

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

5 6 7 8 9 10 11

pH

pH Influences Chymotrypsin Activity

Relative Activity

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

10

9

8

7

6

5

4

3

Buffer pH

pH Influences Net Charge of Protein

Isoelectric point,

pI

+

0

+

-

Net Charge of a Protein

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Imidazole on Histidine Is Affected by pH

H

H

O

C–O-

=

H–O–CH2

C

C

N–H

N

H–N

H–N

H+

C

C

C

C

H

H

H

+

Ser

195

C

N–H

H–N

C

C-H

Asp 102

CH2

His 57

pH 6

pH 7

+

Inactive

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Chymotrypsin Produces New Ile16 N-Terminal

Relative activity

L13

I16

Y146

pH

5 6 7 8 9 10 11

Ile 16

Ile 16

NH2–

Asp 194

–CH2COO-

pH 9

pH 10

+NH3–

New NH2-terminus

pKa

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

New Ile16 N-Terminal Stabilizes Asp194

Catalytic Triad

Gly 193

His 57

Ser 195

Asp 194

Asp 102

+NH3

Ile 16

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Chymotrypsin Ser195 Inhibited by DIFP

O

(CH3)2CH–O–P–O–CH(CH3)2

F

=

O

(CH3)2CH–O–P–O–CH(CH3)2

=

O-…H

CH2

Ser 195

O

CH2

Ser 195

Diisopropyl-fluorophosphate (DIFP)

X

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Addition of Substrate Blocks DIFP Inhibition

100

50

0

Percent Inhibition of activity (%)

No substrate

+ DIFP

X

Add substrate

+ DIFP & substrate

S

Reaction time

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Chymotrypsin Also Catalyzes Acetate

O

CH3–C–O– –NO2

O

-C N-

H

O

CH3–C–OH

O

-C O-

HO– –NO2

Hartley & Kilby

Nitrophenol acetate

+ H2O

Chymotrypsin

Peptide bond

Acetate

Nitrophenol

Ester bond

No acetate was detected at early stage

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Two-Stage Catalysis of Chymotrypsin

O

CH3–C–O– –NO2

O

C

O-

C

O-H

C

O

CH3–C

HO– –NO2

Nitrophenol

Time (sec)

Acylation

Nitrophenol acetate

Kinetics of reaction

Deacylation (slow step)

CH3COOH

+ H2O

Two-phase

reaction

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Extra Negative Charge Was Neutralized

-C-C-N-C-C-N-C-C-N-

H H

O-

-C N-

HOH

O-

-C N-

HOH

O

-C N-

H

O

-C-OH

NH2-

E + S

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Active Site Stabilizes Transition State

Gly 193

Ser 195

Asp 194

Met 192

His 57

Cys 191

Asp 102

Thr 219

Cys 220

Ser 214

Trp 215

Gly 216

Ser 218

Ser 217

Catalytic Triad

Active Site

Specificity Site

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Regulation of Enzyme Activity

Inhibitor

Proteolysis

or

inhibitor

Feedback regulation

Phosophorylation

P

Signal transduction

Regulatory

subunit

(-)

proteolysis

P

R

R

(+)

regulator

effector

phosphorylation

or

+

cAMP or

calmodulin

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Classification of Proteases

2+

Zn

Carboxy-

peptidase A

Non-

polar

EDTA

EGTA

H196

E72

H69

S195-O-

DFP

TLCK

TPCK

Aromatic

Basic

Chymotrypsin

Trypsin

H57

D102

C25-S-

PCMB

Leupeptin

Non-

specific

Papain

H195

D215

Pepsin

Renin

Non-

specific

Pepstatin

H2O

D32

FamilyExampleMechanismSpecificityInhibitor

Metal

Protease

Serine

Protease

Cysteine

Protease

Aspartyl

Protease

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Serine Protease and 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 –

Ser 195

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 – – – – – – – –

His 57

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 – – – – – – –

Asp 102

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

Sigmoidal Curve Effect

vo

ATP

CTP

vo

Noncooperative

(Hyperbolic)

Positive effector (ATP)

brings sigmoidal curve

back to hyperbolic

Negative effector (CTP)

keeps

Sigmoidal curve

Cooperative

(Sigmoidal)

Exaggeration of

sigmoidal curve

yields a drastic

zigzag line that

shows the On/Off

point clearly

Consequently,

Allosteric enzyme

can sense the

concentration of

the environment and

adjust its activity

Off

On

Aulani " Biokimia Enzim " Presentasi 1

[Substrate]


Basic enzyme

Mechanism and Example of Allosteric Effect

Kinetics

Models

Cooperation

vo

(+)

[S]

(+)

vo

(+)

[S]

vo

(-)

(-)

[S]

Allosteric site

R = Relax

(active)

Homotropic

(+)

Concerted

Allosteric site

A

Heterotropic

(+)

Sequential

X

I

Heterotropic

(-)

Concerted

T = Tense

(inactive)

X

X

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

A

A

P

P

P

P

A

A

A

A

P

P

P

P

P

P

A

A

Activity Regulation of Glycogen Phosphorylase

Covalent modification

GP kinase

T

P

T

P

GP phosphatase 1

Non-covalent

ATP

Glc-6-P

Glucose

Caffeine

spontaneously

A

Glucose

Caffeine

AMP

A

R

R

A

Aulani " Biokimia Enzim " Presentasi 1


Basic enzyme

tobe continued ...

thank you

Aulani " Biokimia Enzim " Presentasi 1


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