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

5

Co to jest enzym?

•

Białko

(Struktura 3 i 4

rzędowa)

• Katalizator

(przyspiesza

reakcje)

8

Induced Fit

• A change in the

shape

of an

enzyme’s active

site

• 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

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

Ener

gy
req
uired

(n

o

cat
al

ysi
s)

Ener

gy
decr

eases

(u

nd
er

cat
al

ysi
s)

T = Transition state

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

Active Site Is a Deep Buried Pocket

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

Enzyme Active Site Is Deeper than Binding Site

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

H

O

H

Acid-Base Catalysis

Induced to transition state

C

O

=

N

H

H

C

H

N

H

+

C

-

O

O

H

O

H

-

d

+

d

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

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

Check for

C-terminal

Site for
specificity

Active

site

pocket

Substrate

peptide

chain

R

N

C

N

C

COO

-

O

-

C

+

Zn

Ju

a

n

g

RH

(20

0

4

)

B

Cba

sics

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

Specificity

Site

Catalytic Site

Juang RH (2004) BCbasics

Specificity of Ser-Protease Family

COO

-

C
Asp

Active Site

Trypsin

Chymotrypsin

Elastase

cut at Lys, Arg

cut at Trp, Phe, Tyr

cut at Ala, Gly

Non-polar

pocket

De

ep

and

nega

tiv

ely

ch

ar

ged

poc

ke

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

Ju

a

n

g

RH

(20

0

4

)

B

Cba

sics

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

b

b

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

Ju

a

n

g

RH

(20

0

4

)

B

Cba

sics

1

2

Increa

se
Sub

strate

Concen

trat

ion

2

1

3

4

5

6

7

8

0

0 2

4

6 8

Substrate (

m

mole)

Prod

uc
t

80

60

40

20

0

S

+

E

↓

P

(in a

fixed

period

of

time)

Juang RH (2004) BCbasics

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.

S

E

E

Juang RH (2004) BCbasics

Constant ES Concentration at Steady State

S

P

E

ES

Reaction Time

Conc

en
tra
tio
n

Juang RH (2004) BCbasics

v =

V

max

[S]

K

m

+

[S]

(

v

)

E + S

ES

E + P

k

2

k

1

k

3

For [substrate] low,

k

3

=

k

cat

Problem: Adsorption on solid surfaces has to be avoided!

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

Adsorption on solid surface

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

a

0

20

40

60

80

100

0

3

6

9

pH

a

kt

y

w

n

o

ść

Aktywność enzymu immobilizowanego

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)

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

CH

CH2

COO-

CH

CH2

SO

3

-

+

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.

UF

low ionic strength

high ionic strength

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

Cryogenic transmission electron microscopy (Cryo-TEM)

Localisation of adsorbed protein cont‘d

Ribonuclease A

Bovine hemoglobin

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

„Nanoplant“

Cascade reactions:

Possible system:



-Amylase:

starch



maltose

b

-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

Antibiotics