1

Marek Bryjak

Część II

Zakład Materiałów Polimerowych i Węglowych

Bud. H6, pokój 105

Procesy enzymatyczne

Procesy mikrobiologiczne

Procesy polimeryzacji

Procesy separacyjne (membranowe)

Procesy przygotowania surowców

Procesy wydzielania produktu/ów

Biotechnologia

Inż. chemiczna

Procesy biotechnologiczne

Procesy enzymatyczne

4

Co to jest enzym

Co to jest enzym

?

?

•

Białko

(

(

Struktura 3 i 4

Struktura 3 i 4

rzędowa)

rzędowa)

• Katalizator

(przyspiesza
reakcje)

6

Enzymes

Enzymes

• Are specific for

what they will

catalyze

catalyze

• Are

Reusable

Reusable

• End in –

ase

ase

-

-

Sucrase

Sucrase

-

-

Lactase

Lactase

-

-

Maltase

Maltase

2

8

Induced Fit

Induced Fit

• A change in the

shape

shape

of an

enzyme’s active
site

•

•

Induced

Induced

by the

substrate

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

The Nature of Enzyme Catalysis

●

●

Enzyme provides a catalytic surface

Enzyme provides a catalytic surface

●

●

This surface stabilizes transition state

This surface stabilizes transition state

●

●

Transformed transition state to product

Transformed transition state to product

B

B

A

Catalytic surface

A

Juang RH (2004) BCbasics

Enzyme Stabilizes Transition State

S

P

ES

ES

T

EP

S

T

Reaction direction

Energy change

E

n

er

g

y

r

eq

u

ir

ed

(

n

o

c

at

al

ys

is

)

E

n

er

g

y

d

ec

re

as

es

(

u

n

d

er

c

at

al

ys

is

)

What’s the difference?

T = Transition state

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

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

+

-

Juang RH (2004) BCbasics

3

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.

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

X

H

O

H

Acid-Base Catalysis

A

d

a

p

te

d

f

ro

m

N

e

ls

o

n

&

C

o

x

(2

0

0

0

)

L

e

h

n

in

g

e

r

P

ri

n

c

ip

le

s

o

f

B

io

c

h

e

m

is

tr

y

(

3

e

)

p

.2

5

2

Induced to transition state

C

O

=

N

H

H

C

H

N

H

+

C

-

O

O

H

O

H

-δ

+δ

H

O

H

C

O

=

N

H

H

C

H

C

O

=

N

H

H

C

H

C

O

=

N

H

H

C

H

Slow

Fast

Fast

Very Fast

Acid-base

Catalysis

Base

catalysis

Acid

catalysis

Both

A

d

a

p

te

d

f

ro

m

A

lb

e

rt

s

e

t

a

l

(2

0

0

2

)

M

o

le

c

u

la

r

B

io

lo

g

y

o

f

th

e

C

e

ll

(4

e

)

p

.1

6

7

N

H

+

C

-

O

O

H

O

H

Specific

Concerted Mechanism of Catalysis

1

2

3

4

5

O

-

H

+

H

COO

-

(270)

Glu

(248)

Tyr

O

-

H

His

(196)

His (69)

Glu

(72)

+

Arg (145)

Carboxypeptidase A

C-terminus

ACTIVE

SITE

ACTIVE

SITE

Check for
C-terminal

Site for
specificity

Active

site

pocket

Substrate

peptide

chain

R

N

C

N

C

COO

-

O

-

C

+

Zn

J

u

a

n

g

R

H

(

2

0

0

4

)

B

C

b

a

s

ic

s

O

O

N–C–C–

N–C–C

N–C–C

–N–C–C

R

H

R’

Chymotrypsin Has A Site for Specificity

O

-

C

Ser

Active Site

Active Site

Specificity

Site

Specificity

Site

Catalytic Site

Juang RH (2004) BCbasics

Asp102

His57

Ser195

Catalytic Triad

Catalytic Triad

H

H

Chymotrypsin Catalytic Mechanism A1

N

C

C

N

[

HOOC]

H

O

C

C

N

C

C

[

NH

2

]

C

C

O

Check substrate specificity

Asp102

His57

Ser195

H

H

Chymotrypsin Catalytic Mechanism A2

N

C

C

N

[HOOC]

H

O

C

C

N

C

C

[NH

2

]

C

C

O

First Transition State

4

H

H

Chymotrypsin Catalytic Mechanism A3

N

C

C

N

[HOOC]

H

O

C

C

N

C

C

[NH

2

]

C

C

O

Acyl-Enzyme Intermediate

H

Chymotrypsin Catalytic Mechanism D1

N

-H

C

C

N

[HOOC]

H

O

C

C

N

C

C

[NH

2

]

C

C

O

H

O

H

Acyl-Enzyme Water Intermediate

H

Chymotrypsin Catalytic Mechanism D2

O

O

C

C

N

C

C

[NH

2

]

C

C

H

Second Transition State

O

H

H

Chymotrypsin Catalytic Mechanism D3

O

C

C

N

C

C

[

NH

2

]

C

C

O

O

H

Deacylation

H

Specificity of Ser-Protease Family

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-polar

pocket

D

e

e

p

a

n

d

n

e

g

a

ti

v

e

ly

c

h

a

rg

e

d

p

o

c

k

e

t

Shallow and

non-polar

pocket

O O

–C–

N–C–C

–

N–

C
C
C
C
NH

3

+

O O

–C–

N–C–C

–

N–

C

O O

–C–

N–C–C

–

N–

CH

3

J

u

a

n

g

R

H

(

2

0

0

4

)

B

C

b

a

s

ic

s

24

Two examples of Enzyme

Two examples of Enzyme

Inhibitors

Inhibitors

a.

a.

Competitive

Competitive

inhibitors

inhibitors

:

:

are chemicals
that

resemble

resemble

an

enzyme

enzyme

’

’

s

s

normal

normal

substrate

substrate

and

compete

compete

with

it for the

active site

active site.

Enzyme

Competitive inhibitor

Substrate

5

25

Inhibitors

Inhibitors

b.

b.

Noncompetitive

Noncompetitive

inhibitors

inhibitors

:

:

Inhibitors that

do

do

not enter the

not enter the

active

active

site

site,

but

bind to

bind to

another part

another part

of the

enzyme

enzyme

causing the

enzyme

enzyme

to

change its

change its

shape

shape

, which in turn

alters the active

alters the active

site

site.

Enzyme

active site

altered

Noncompetitive

Inhibitor

Substrate

Invertase (IT)

IT

Sucrose

Non-reducing sugar

Reducing

sugars

Glucose

Fructose

Reducing Power

+

HOCH

2

O

OH

1

2

3

4

5

6

6

5

4

3

2

1

1

2

3

4

5

6

HOCH

2

O

OH

O

HOCH

2

HOCH

2

OH

H

2

O

O

HOCH

2

HOCH

2

HO

O

HOCH

2

O

HOCH

2

HOCH

2

O

β

β

CHO

H-C-OH

HO-C-H

H-C-OH

H-C-

OH

H

2

-C-OH

H

2

C-OH

C=O

HO-C-H

H-C-OH

H-C-

OH

H

2

-C-OH

J

u

a

n

g

R

H

(

2

0

0

4

)

B

C

b

a

s

ic

s

1

2

In

cr

e

a

se
S

u

b

s

tra

te

C

o

n

ce
n

tra

tio

n

2

1

3

4

5

6

7

8

0

0 2 4 6 8

Substrate (µmole)

P

ro

d

u

ct

80

60

40

20

0

S

+

E

↓

P

(in

a

fix

ed

p

er

io

d o

f ti

m

e)

Juang RH (2004) BCbasics

Essential of Enzyme Kinetics

E

S

+

P

+

Steady State Theory

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.

S

E

E

Juang RH (2004) BCbasics

Constant ES Concentration at Steady State

S

P

E

ES

Reaction Time

C

o

n

ce
n

tra

tio

n

Juang RH (2004) BCbasics

6

v

o

=

V

max

[S]

K

m

+

[S]

(

v

o

)

E + S

ES

E + P

k

2

k

1

k

3

For [substrate] low,

k

3

=

k

cat

7

Problem: Adsorption on solid surfaces has to be avoided!

M. Santore et al., Langmuir 2002, 18 (3), 706.

Adsorption on solid surface

Denaturation of proteins
by adsorption on solid
surfaces

- strong attraction by

van der Waals or
hydrophobic interaction

⇒

Loss of biological
function

8

Czynniki wiążące

•

Gr funk nośnika

Gr funk białka

Czynniki wiążący

•

-COOH

-NH

2

karbodiimid

•

-COOH

- -COOH

izocyjanki

•

-COOH

-CH

2

, -SH

azydek

•

-OH

NH

2

bromocyjan

•

-NH

2

-NH

2

aldehyd glutarowy

•

-NH

2

-COOH

karbodimid

•

-NH

2

-COOH

izocyjanki

•

-NH

2

--NH

2

triazol

•

Grupy oksiranowe

-NH

2

, -OH

•

-OH

- -NH

2

, -OH

diwinylosulfon

•

-OH

NH

2

hydrazyna

•

-OH

NH

2

karbodiimid

•

-OH

NH

2

nadjodan sodu

Aktywność immobilizowanego enzymu

Enzym sieciowany i kryształy

a

0

20

40

60

80

100

0

3

6

9

pH

a

k

ty

w

n

o

ść

Aktywność enzymu immobilizowanego

9

Immobilizacja enzymów w objętości bioreaktora

inkludowanie

związanie z nośnikiem

sieciowanie

-

w sieci żelu

- adsorpcyjnie

rozpuszczonego białka -

- otoczkowanie

- koordynacyjnie

- agregatów białkowych

- w mikro(makro)kapsułkach

- specyficznie

- kryształów białka

- w mikro(makro)emulsjach

- kowalencyjnie

-(w materiale membrany)

- (

na powierzchni membrany)

Nierozpuszczalne w wodzie

Przykład

Immobilization of proteins on colloidal carriers

„bionanoparticles“

Colloidal particles

• provide large surfaces

• large amount of immobilized

biomolecules

Enzymes can be used
as catalysts for
technical applications

substrate

bound
enzymes

product

PS

R

L

CH

CH2

COO-

CH

CH2

SO

3

-

•

Long charged polyelectrolytes attached to colloidal particles

weak
polyelectrolyte

strong
polyelectrolyte

Can be used as
carrier particles
for proteins

Spherical Polyelectrolyte Brush (SPB)

10

Confinement of counterions inside brush layer

PS

R

L

CH

CH 2

SO

3

-

Confined counterions

• high osmotic pressure inside brush

• chains strongly stretched

⇒

Properties of the particles determined

by the confinement of the counterions

PS

R

L

+

negatively charged

negatively charged

carrier

protein

Adsorption on the

„wrong side“: pH > pI

Double trouble:

Electrostatic repulsion + steric
repulsion

? ?

Protein adsorption on Spherical Polyelectrolyte Brushes ?

Protein adsorption: Experimental procedure

Wittemann et al., Phys. Chem. Chem. Phys. 2003, 5, 1671.

Ultra-

filtration

PS

PS

PS

Mixing

Protein solution

Brush latex

Protein coated
brush latex

Protein coated brush
latex + dissolved
proteins

A certain amount of protein remains
adsorbed after exhaustive ultrafiltration!

Main driving force:

Counterion release force

Uptake of protein leads to release of counterions

• strong driving force for protein adsorption even at „wrong“

side of the IEP

High
osmotic
pressure
partially
relieved by
multi-valent
counterions

+
+

+

+

-

-

-

+

+

+

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

--

--

N

+

N

-

-

-

-

+

+

+

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+
+

+

+

+

+

+

--

--

2N

+

- N

-

released counterions

+

+
+

+

+

+

+

+

--

--

--

+

+

+

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

--

--

--

--

--

--

--

--

--

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

--

--

N

+

N

-

--

--

--

+

+

+

+

+

+

--

--

--

--

--

--

--

--

--

+

+

+

+

+

+

+

+

+

+

+

+

+
+

+

+

+

+

+

--

--

+

+
+

+

+

+

+

+

+

+

+

--

--

+

+

+

--

--

2N

+

- N

-

released counterions

Polyelectrolyte Mediated Protein Adsorption (PMPA)

Review on the PMPA:

Wittemann, A.; Ballauff, M.
Phys. Chem. Chem. Phys. 2006.

Theoretical description:

Leermakers, F.A.M.; Ballauff,
M.; Borisov, O.V.

Langmuir, in

press.

low ionic strength

high ionic strength

Wittemann et al., J. Am. Chem. Soc. 2005, 127, 9688.

Cryogenic transmission electron microscopy (Cryo-TEM)

Osmotic brush:
confined counterions,

c

s

>

c

a

Salted brush: c

s

=

c

a

11

Localisation of adsorbed protein cont‘d

Ribonulease A

Bovine hemoglobin

Adsorption onto SPB consisting of strong polyelectrolytes

Activity of
enzyme preserved

[ ]

[ ]

S

K

S

v

v

M

+

=

max

0

Activity of bound glucoamylase: Michaelis-Menten analysis

Neumann et al., Macromol. Biosci. 2004, 4, 13; Haupt et al., Biomacromolecules 2005, 6, 948.

-1/K

m

PS

Aim:

„Nanoplant“

Cascade reactions:

Possible system:

α

-Amylase:

starch → maltose

β

-Glucosidase:

maltose → glucose

Glucose Oxidase:

glucose → H

2

O

2

enzyme A

enzyme B

end product

PS

R

L

CH

CH2

COO-

CH

CH2

SO

3

-

protein

„Nanoreactor“

Carrier
particles
for
proteins

Confined counterions

Polyelectrolyte-

protein complexes

Polyelectrolyte-mediated

protein adsorption

• both can take place on the „wrong side“ of the IEP

• both can be traced back to patches of opposite charge

However:

PMPA

leads to

stronger protein binding

because

of the

Donnan effect

and a much

stronger correlation of

the counterions

Synthesis: photoemulsion polymerization

Guo et al., Macromolecules 1999, 32, 6043.

r.t., h

ν

acrylic
acid

PS

PAA

PS

photo-
initiator

70°C

PS

Step 1:

PS latex

Step 2

Photoinitiator layer

Step 3

Shell composed of
linear polyelectrolytes

12

SPB: decisive parameters

R:

core radius

L:

hydrodynamic brush
layer thickness

L

C

:

contour length of the
poly(acrylic acid) chains

σ

σ

σ

σ

:

grafting density of the
tethered chains

D:

average distance of the
grafting points

PS

PAA

R

L

C

D

L

Guo et al., Langmuir 2000, 16, 8719.

id

Π

Π

=

φ

kT

n

R

id

=

Π

Osmotic coefficient φ: Fraction of „free“ counterions

Measurement of
the osmotic
pressure of dilute
salt-free
suspensions

ca. 95 % of
counterions
confined in brush
as predicted for
osmotic limit

water

membrane

∆

p <

0

pressure

solution

Antibiotics

13