Sie sind auf Seite 1von 40

Bioprocess Engineering

ENZYME CATALYSIS
Definition
Essential macromolecules that have a key
role in the catalyzing the chemical
transformations that occur in all living
systems metabolisms
The nature and specifity of their catalytic
activity is primarily due to the three
dimensional structure of the folded protein
which is determined by the sequence of the
amino acids that make up the enzyme and the
activity may be regulated by one ore more
small non protein molecules (cofactors)
which cause small conformation changes in
the enzyme structure.
Cofactor type consit of metal ions and organic molecules (co-enzyme)
Apoenzyme : catalytically inactive enzyme
Haloenzyme : active enzyme that tightly bounded cofactors
Haloenzyme
cofactor
Inactive site
Active Site
ENZYME MECHANISM
Apoenzyme
Enzyme Classification
Oxidoreductase, catalyze the transfer of hydrogen or
oxygen atoms or electrons from one substrate to another
(alcohol dehydrogenase, fatty acid desaturase, glucose
oxidase, peroxidase)
Transferase, catalyze the group transfer reactions
(aspartase, transaminase, dextransucrase,
phosphorylase)
Hydrolases, catalyze the hydrolitic reactions
(amylase,lipase, protease, cellulase, isoamylase, urease,
papain)
Lyases, catalyze the non hydrolytic removal of groups
from substrate (lactase, tannase, aspartase,
tryptophanase)
Isomerase, catalyze isomerization reactions (glucose
isomerase)
Ligases, catalyze the synthesis of various type of bonds
where the reaction are coupled with breakdown of
energy containig materials (microbial enzyme,
carbamate kinase)
Enzyme Activity
Enzyme are specific in function; the reaction of an
enzyme with certain substrate involves the
formation of an intermediate which then reacts
further with another substrates or decompose to
form products.
Activity is given by the amount of product formed
or substrate consumed in the reaction mixture
under specified conditions ( T, pH, buffer type,
substrate)
Enzyme concentration was defined as the amount
of enzyme which gives a certain amount of catalytic
activity under specified condition
ion concentrat enzyme
activity
activity specific =
ml
enzyme mmole
ml
product mmole
min
Kinetics of Single Substrate Reactions
L. Michaelis and M. L. Menten (1913)
ES S E
k
k

+
1
1
E P ES
k
+

2
| | ES E E + =
0
| |
| | ( ) | |
| | | | ( ) ES E S
k k
k
E S
k k
k
ES
ES k k E S k ES
ES
dt
d
dt
d

+
=
+
=
+ =
=

0
2 1
1
2 1
1
2 1 1
0
| | ; 0
0
= ES
| |
( )
| |
M
dt
d
dt
d
K S
S P
k
k k
S
S E k
ES k P
+

=
|
|
.
|

\
|
+
+

= =

max
1
2 1
0 2
2
Michaelis - Menten Mechanism
Derivatived Equation of Michalis-Menten Mechanism
| |
S
k
k k
S E
S
k k
k
S E
k k
k
ES
+
+

=

+
+

+
=

1
2 1
0
2 1
1
0
2 1
1
1
Haloenzyme
Inactive site
Active Site
Apoenzyme
substrate
cofactor
product
MECHANISM SCHEME
W.P. Jenks and J. Carriuolo, 1961
Jenks-Carriuolo Mechanisms
Derivatived Equation of Jenks-Carriuolo Mechanism
ES S E
k
k

+
1
1
E P ES
k
+

2
'
' ES ES
p
p
k
k

| | | | ; 0 ' ,
0 0
= ES ES
| | | | '
0
ES ES E E + + =
| | | |
| | | | ( ) | |
| | | | ( ) | | ' '
'
0 ' ,
2
1 1
ES k k ES k ES
ES k k ES k E S k ES
ES ES
p p dt
d
p p dt
d
dt
d
dt
d
+ =
+ + =
=


| |
( )
| |
| | ( ) | | 0
'
1
2
1
2
= +
|
|
.
|

\
|

+
+

+
=

ES k k ES
k k
k
k E S k
ES
k k
k
ES
p
p
p
p
p
p
| | | | ( ) | | ( ) | | 0 '
1
2
1 0
= +
|
|
.
|

\
|

+
+

ES k k ES
k k
k
k S k ES ES E
p
p
p
p
Haloenzyme
Inactive I site
inactive II Site
Apoenzyme
substrate
cofactor
product
MECHANISM SCHEME
Active Site
substrate
| | | | | | ( ) | |
| | | | ( ) | | | |
| |
( )
| |
( )
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+
+
+

|
|
.
|

\
|
+
+

=
|
|
.
|

\
|
+
+ +
|
|
.
|

\
|
+
+

=
|
|
.
|

\
|

+
+ +
|
|
.
|

\
|

+
+ =
= +
|
|
.
|

\
|

+
+
|
|
.
|

\
|

2
1
2
1
2
1
0 1
2
1
2
1
0 1
2
1
2
1 0 1
1
2
1
2
0
1
1
1
0
k k
k
k
k k
k
k k k
S
S
k k
k
k
E k
ES
k k
k
k k k S
k k
k
k
E S k
ES
ES
k k
k
k ES k k ES
k k
k
ES S k E S k
ES k k ES
k k
k
k S k ES
k k
k
ES E
p
p
p
p
p p
p
p
p
p
p p
p
p
p
p
p p
p
p
p
p
p
p
p
p
| |
( )
| | ES
k k
k k
ES k P
dt
d
p
p

= =
2
2
2
'
( )
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+
+
+

|
|
.
|

\
|
+
+

2
1
2
1
2
0
2
2
1
1
k k
k
k
k k
k
k k k
S
S
k k
k
E
k k
k k
P
dt
d
p
p
p
p
p p
p
p
p
p
( ) ( )
( ) ( )
( )
|
|
.
|

\
|
+
+
+
+ + +
+

|
|
|
|
|
.
|

\
|

+
+
+
=
|
|
.
|

\
|
+
+
+
+ +
+

|
|
|
|
|
.
|

\
|

+
+
+
=
|
|
.
|

\
|
+
+
+
+ +
+

|
|
|
|
|
.
|

\
|

+
+
+
=

2
1
2
2 2 1 1
0 2
2
2
2
1
2
2 1
0 2
2
2
2
1
2
2 1
0 2
2
2
1
1
1
1
1
1
k k
k
k
k k
k k k k k k k k k k
S
S
E k
k k
k
k k
k
P
dt
d
k k
k
k
k k
k k k k k k
S
S
E k
k k
k
k k
k
P
dt
d
k k
k
k
k k
k k k k k k
S
S
E k
k k
k
k k
k
P
dt
d
p
p
p
p p p p p p
p
p
p
p
p
p
p
p p p p
p
p
p
p
p
p
p
p p p p
p
p
p
p
( )
|
|
|
|
|
.
|

\
|
+

+
+

+
+

|
|
|
|
|
.
|

\
|

+
+
+
=
|
|
.
|

\
|
+
+
+
+ +
+

|
|
|
|
|
.
|

\
|

+
+
+
=

2
1
1
2
2
1
0 2
2
2
2
1
2
2 2 1
0 2
2
2
1
1
1
k k
k k
k
k k
k k
k
S
S
E k
k k
k
k k
k
P
dt
d
k k
k
k
k k
k k k k k
S
S
E k
k k
k
k k
k
P
dt
d
p
p
p
p
p
p
p
p
p
p
p
p p
p
p
p
p
|
|
|
|
|
.
|

\
|
+

+
+

+
=
|
|
|
|
|
.
|

\
|

+
+
+
=
(

2
1
1
2
2
1
0 2
2
2
max
max
1
:
k k
k k
k
k k
k k
k
K dan E k
k k
k
k k
k
P
dt
d
here
K S
S
P
dt
d
P
dt
d
p
p
p
p
M
p
p
p
p
M
Briggs-Haldane (1925)
Briggs-Haldane Mechanism
Derivatived Equation of Briggs-Haldane Mechanism
ES S E
k
k

+
1
1
E P ES
k
+

2
"
" ' ES ES ES
q
q
p
p
k
k
k
k


| | | | | | ; 0 " , ' ,
0 0
= ES ES ES | | | | | | " '
0
ES ES ES E E + + + =
| | | | | |
| | | | ( ) | |
| | | | | |
| | | | ( ) | | " ' "
' '
'
0 " , ' ,
2
1 1
ES k k ES k ES
ES k ES k ES
ES k k ES k E S k ES
ES ES ES
q q dt
d
p p dt
d
p p dt
d
dt
d
dt
d
dt
d
+ =
=
+ + =
=


| |
( )
| |
| | | |
| | ( ) | |
| |
| |
| | | | | | | | ( ) | | " '
0
0
'
' "
0
1
1
1
1
1 1
1 1
2
ES ES ES E S
k
k
ES
E S
k
k
ES
ES k E S k
ES k k ES
k
k
k E S k
ES
k
k
ES
ES
k k
k
ES
p
p
p
p
p
p
q
q
+ + =
=
=
= +
|
|
.
|

\
|
+
=

+
=

| |
( )
| |
( )
| |
| | | | | |
( )
| |
(
(

|
|
.
|

\
|
|
|
.
|

\
|

+
+
|
|
.
|

\
|
+ =

+
=
|
|
.
|

\
|

+
=


ES
k k
k
k
k
ES
k
k
ES E S
k
k
ES
ES
k k
k
k
k
ES
k
k
k k
k
ES
q
q
p
p
p
p
q
q
p
p
p
p
q
q
2
0
1
1
2 2
"
| |
( )
| |
( )
| |
( )
( )
( )

|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+

|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+

|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+ +

|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+ +

=
=

|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+ +



S
k
k
k k
k
k
k
k
k
k k
k
k
k
k
k
E S
S
k k
k
k
k
k
k
k
k
E S
ES
k k
k
k
k
k
k
S
k
k
E S
k
k
ES
E S
k
k
k k
k
k
k
k
k
S
k
k
ES
q
q
p
p
p
p
q
q
p
p
p
p
q
q
p
p
p
p
q
q
p
p
p
p
q
q
p
p
p
p
1
1
2
2
0
2
1
1
0
2 1
1
0
1
1
0
1
1
2 1
1
1
1
1
1
1
1 1
1 1
| |
( )
| |
( )
| | ES
k k
k k
k
k
ES
k
k
k k
k
k ES k P
dt
d
q
q
p
p
p
p
q
q

=
|
|
.
|

\
|

+
= =
2
2
2
2 2
"
( )
| |
( )
( )
( )

|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+

|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+
+

=
+

=




S
k
k
k k
k
k
k
k
k
E S
k k
k
k
k
k
k
k k
k k
k
k
ES
k k
k k
k
k
P
dt
d
q
q
p
p
p
p
q
q
p
p
p
p
q
q
p
p
q
q
p
p
1
1
2
0
2
2
2
2
2
1
1
1
( )
( )
( )
S
k k
k
k
k
k
k
k
k
S
k k
k
k
k
k
k
E
k k
k k
k
k
P
dt
d
q
q
p
p
p
p
q
q
p
p
p
p
q
q
p
p
+
|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+

|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+



2
1
1
2
0
2
2
1
1
S K
S P
dt
d
P
dt
d
M
+

=
max
( )
( ) ( )
|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+
=
|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+

=
(



2
1
1
2
0
2
2
max
1
,
1
:
k k
k
k
k
k
k
k
k
K dan
k k
k
k
k
k
k
E
k k
k k
k
k
P
dt
d
here
q
q
p
p
p
p
M
q
q
p
p
p
p
q
q
p
p
0
max
E k P
dt
d
cat
=
(

Turnover Number (k
cat
)
Catalytic center activity rate constant
The maximum number of substrate molecules that can be converted to product
per unit time per active site on the enzyme (S >> K
M
)
Jenks-Carriuolo
Michaelis-Menten
2
k k
cat
=
2
2
2
1
k
k k
k
k k
k
k
p
p
p
p
cat

|
|
|
|
|
.
|

\
|
+
+
+
=

( )
( )
2
2
2
1
k
k k
k
k
k
k
k
k k
k
k
k
k
q
q
p
p
p
p
q
q
p
p
cat

|
|
|
|
|
.
|

\
|
|
|
.
|

\
|
+
+
|
|
.
|

\
|
+
+

=


Briggs-Haldane
In case of k
-p
was assumed very small, then:
Jenks-Carriuolo
Michaelis-Menten
2
2 2
2
2
2
1
1
1
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
q p
p
q
q
p
p
p
p
q
p
p
cat

|
|
|
|
|
.
|

\
|
|
|
.
|

\
|

|
|
.
|

\
|
+
|
|
.
|

\
|
+
=
|
|
|
|
|
.
|

\
|
|
|
.
|

\
|

|
|
.
|

\
|
+
|
|
.
|

\
|
+
|
|
.
|

\
|

|
|
.
|

\
|
=

Briggs-Haldane
In case of k
-q
was assumed very small, then:
If k
-p
and k
q
was equal, then:
|
|
.
|

\
|
= =
2
2
1
1
k
k k
cat
|
|
.
|

\
|
+ +
=
|
|
|
|
|
.
|

\
|
|
|
.
|

\
|
+
|
|
.
|

\
|
+
=
p q p q
cat
k k k
k
k
k
k
k
k
1 1 1
1
1
1
2
2
2 2
|
|
.
|

\
|
+
=
|
|
|
|
.
|

\
|
+
=
|
|
|
|
.
|

\
|
+
=
p
p
p
p
cat
k k
k
k
k
k
k
k
k
k
k
1 1
1
1
1
1
2
2
2
2
2
2
Multiple intermediete Mechanism
Turnover Number
|
|
.
|

\
|
+ + + + +
=
p q r z
cat
k k k k k
k
1 1 1
...
1 1
1
2
E P ES ES ES ES S E
k
z
z
k
r
k
q
k
p
k
p
k
k
k
+

+

2 1
1
... " '
J. F. Andrews (1968)
Inhibitory Substrate Mechanisms
ES S E
k
k

+
1
1
2
2
2
ES S ES
k
k

+ E P ES
k
+

3
Derivatived Equation of Inhibitory Substrate Mechanism
| | | | ; 0 ,
0 2 0
= ES ES | | | |
2 0
ES ES E E + + =
| | | |
| | | | | | | | | |
| | | | | |
| | | |
| | | | | | | |
| | | | | | ( )
2 0
3 1
1
3 1
1
2
2
2 2 3 1 1
2
2
2
2 2 2 2
2 2 2 3 1 1
2
0
0 ; 0
ES ES E S
k k
k
E S
k k
k
ES
S ES
k
k
k S ES k ES k ES k E S k
S ES
k
k
ES
ES k S ES k ES
ES k S ES k ES k ES k E S k ES
ES ES
dt
d
dt
d
dt
d
dt
d

+
=
+
=
=
|
|
.
|

\
|
+
=
=
+ =
= =


Haloenzyme
Inactive site
Active Site
Apoenzyme
substrate
cofactor
product
MECHANISM SCHEME
Inactive Site
| | | |
| | | | | |
| | | | | |
| |
| |
| |
| |
S
k
k
S k
k k
E
ES
S
k
k
S k
k k
E
ES
S
k k
k
k
k
S
k k
k
E S
k k
k
ES
E S
k k
k
S
k k
k
S
k k
k
k
k
ES
E S
k k
k
ES S ES
k
k
S
k k
k
ES
ES S ES
k
k
E S
k k
k
ES
S ES
k
k
ES
+
+
+
=
+ +
+
=
+
+
+
+

+
=

+
=
)
`

+
+
+
+

+
=
|
|
.
|

\
|
+
+
+
|
|
.
|

\
|

+
=
=

2
2
1
3 1
0
2
2
1
3 1
0
2
3 1
1
2
2
3 1
1
0
3 1
1
0
3 1
1
3 1
1
2
3 1
1
2
2
0
3 1
1
2
2
3 1
1
2
2
0
3 1
1
2
2
2
1
1
1
1
1
1
| |
2
2
,
1
3 1
, 0 3
max
,
,
max
1
3 1
0
3 3
; ;
:
1
1
1
k
k
K
k
k k
K E k P
dt
d
here
K
S
S
K
P
dt
d
P
dt
d
S
k
k
S k
k k
E
k ES k P
dt
d
s I s A
s I
s A
i
i

=
+
= =
(

+ +
(

=
+
+
+
= =
In case of high value of S above equation become then:
s I
K
S
P
dt
d
P
dt
d
,
max
1+
(

=
In case of low value of S above equation become then:
S K
S P
dt
d
S
K
P
dt
d
P
dt
d
M
s A
+

~
+
(

=
max
,
max
1
Multisite Enzyme Kinetics
Question
Suppose that an enzyme has two active sites so that substrate is
converted to product via the reaction sequence:
Derive a rate expression for P formation by assumming quasy
steady state for (ES) and for (ESS).
P E ES
P ES ESS
ESS S ES
ES S E
k
k
k
k
k
k
+
+
+
+

4
3
2
2
1
1
) (
) ( ) (
) ( ) (
) (
Problems (G.F. Webb 1986)
Answer:
quasy steady state for (ES) and for (ESS).
0 ) (
0 ) (
=
=
ESS
ES
dt
d
dt
d
and then,
( ) ( ) ( ) 0
3 2 2
=

ESS k ESS k ES k
( ) | | ( ) 0
3 2 2
= +

ESS k k ES k
( ) | | ( ) ESS k k ES k + =
3 2 2
( ) ( ) ES
k k
S k
ESS
+
=
3 2
2
( ) ( ) ( ) ( ) 0
4 2 2 1 1
= +

ES k ESS k S ES k ES k ES k
( )( ) ( ) ( )( ) 0
3 2 2 4 1 1
= + + +

ESS k k S ES k ES k k ES k
( )( ) ( ) ( )
( )
0
3 2
2
3 2 2 4 1 1
=
+
+ + +


k k
S ES k
k k S ES k ES k k ES k
( )( ) 0
4 1 1
= +

ES k k ES k
( )
4 1
1
k k
ES k
ES
+
=

( ) ( ) ( ) ( ) ES
k k
S k
ES E ESS ES E E
o
3 2
2
+
+ + = + + =

( )
|
|
.
|

\
|
+

|
|
.
|

\
|
+
+ + =
|
|
.
|

\
|
+
+ + =
4 1
1
3 2
2
3 2
2
1 1
k k
ES k
k k
S k
E ES
k k
S k
E E
o
|
|
.
|

\
|
|
|
.
|

\
|
+

|
|
.
|

\
|
+
+ + =
4 1
1
3 2
2
1 1
k k
S k
k k
S k
E E
o
|
|
.
|

\
|
|
|
.
|

\
|
+

|
|
.
|

\
|
+
+ +
=
4 1
1
3 2
2
1 1
k k
S k
k k
S k
E
E
o
( )
|
|
.
|

\
|
|
|
.
|

\
|
+

|
|
.
|

\
|
+
+ +

+
=

4 1
1
3 2
2
4 1
1
1 1
k k
S k
k k
S k
E
k k
S k
ES
o
( ) ( ) ES
k k
S k
ESS
+
=
3 2
2
( )
(
(
(
(
(

|
|
.
|

\
|
|
|
.
|

\
|
+

|
|
.
|

\
|
+
+ +

+
|
|
.
|

\
|
+
=


4 1
1
3 2
2
4 1
1
3 2
2
1 1
k k
S k
k k
S k
E
k k
S k
k k
S k
ESS
o
derivative rate expression for P formation:
( ) ( ) ES k ESS k P
dt
d
4 3
+ =
|
|
.
|

\
|
+
+
|
|
.
|

\
|
|
|
.
|

\
|
+

|
|
.
|

\
|
+
+ +

+
=


3 2
2
3 4
4 1
1
3 2
2
4 1
1
1 1
k k
S k
k k
k k
S k
k k
S k
E
k k
S k
P
o
dt
d
if:
4
3
2
3 2
,
1
4 1
,
, ,
k
k
k
k k
K
k
k k
K
s I s A
=
+
=
+
=

|
then:
|
|
.
|

\
|
+
|
|
.
|

\
|
|
|
.
|

\
|

|
|
.
|

\
|
+ +
=
s I
s A s I
o
s A
dt
d
K
S
K
S
K
S
E k
K
S
P
,
, ,
4
,
1
1 1
|
,
| |
o
dt
d
E k P
4
max
=
| |
|
|
.
|

\
|
+
|
|
.
|

\
|
+ +
=
|
|
.
|

\
|
+
|
|
.
|

\
|
+ +
=
s I
s I
s A
dt
d
s I
s I s A s A
o
s A
dt
d
K
S
K
S
S
K
P
K
S
K
S
K
S
K
S
E k
K
S
P
,
,
,
max
,
, , ,
4
,
1
1
1
1
| |
derivative rate expression for P formation:
| |
+ +
+
=
s I
s A
s I
dt
d
dt
d
K
S
S
K
K
S
P P
,
,
,
max
1
1 |
:
| |
+ +
+
=
s I
s A
s I
dt
d
dt
d
K
S
S
K
K
S
P P
,
,
,
max
1
1 |
in case
1 = |
, then
| |
+ +
+
=
s I
s A
s I
dt
d
dt
d
K
S
S
K
K
S
P P
,
,
,
max
1
1
(Webb Equation)
Name Kinetics (dP/dt)
Yano
Ierusalimsky
Chen
Substrate uptake kinetics including inhibition
|
|
.
|

\
|
|
|
.
|

\
|
+ + +

2
, 2 , 1
,
max
1
s I s I
s A
K
S
K
S
S K
S
P
dt
d
|
|
.
|

\
|
+

s I
s A
K
S
S K
S
P
dt
d
,
, max
1
1
( )
2
, 2 , 1 , max
1
S K S K
S
K
S
P
dt
d
s A s A s I
+
|
|
.
|

\
|

(

Competitive Inhibition
Inhibitory Mechanisms
ES S E
k
k

+
1
1
EI I E
i
i
k
k

+
E P ES
k
+

2
Derivatived Equation of Inhibitory Substrate Mechanism
| | | | ; 0 ,
0 0
= EI ES | | | | EI ES E E + + =
0
| | | |
| | | | | | | |
| | | |
| |
| | | |
| | | | | | ( ) EI ES E S
k k
k
E S
k k
k
ES
I E
k
k
k I E k ES k ES k E S k
I E
k
k
EI
EI k I E k EI
EI k I E k ES k ES k E S k ES
EI ES
i
i
i i
i
i
i i dt
d
i i dt
d
dt
d
dt
d

+
=
+
=
=
|
|
.
|

\
|
+
=
=
+ =
= =


0
2 1
1
2 1
1
2 1 1
2 1 1
0
0 ; 0
| | | |
| |
I
k
k
ES E
E ES I
k
k
E I E
k
k
ES E E
i
i
i
i
i
i
+

= +
|
|
.
|

\
|
+ = + + =


1
, 1
0
0
Haloenzyme
Inactive site
Active Site
Apoenzyme
substrate
inhibitor
cofactor
product
MECHANISM SCHEME
| |
| |
| |
| |
| |
| |
| |
I
k
k
S
k k
k
I
k
k
E
S
k k
k
ES
I
k
k
E
S
k k
k
I
k
k
S
k k
k
ES
I
k
k
E
S
k k
k
I
k
k
ES
S
k k
k
ES
I
k
k
ES E
S
k k
k
ES
I
k
k
ES E
E
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
+

+
+
+

+
=
+

+
=

+
+
+

+
=
|
|
|
|
.
|

\
|
+

+
+
|
|
|
|
.
|

\
|
+


+
=
+

1
1
1
1 1
1
1 1
1
1
2 1
1
0
2 1
1
0
2 1
1
2 1
1
0
2 1
1
2 1
1
0
2 1
1
0
| |
| |
| |
| |
| |
| |
( )
|
|
.
|

\
|
+
|
|
.
|

\
| +
+

= =
|
|
.
|

\
|
+
|
|
.
|

\
| +
+
=
+
|
|
.
|

\
|

+
+
+
=
+
+

|
|
.
|

\
|
+

=
|
|
.
|

\
|
+
+

|
|
.
|

\
|
+
+

+

+

+
=
|
|
.
|

\
|
+
+

|
|
.
|

\
|
+
+

+

+

+
=

I
k
k
S k
k k
E k
ES k P
dt
d
I
k
k
S k
k k
E
ES
S
I
k
k k
k
k
k
k k
E
ES
S
k
k k
I
k
k
S E
ES
S
k
k k
I
k
k
I
k
k
k k
k
S E
I
k
k
k k
k
ES
S
k
k k
I
k
k
I
k
k
k k
k
S E
I
k
k
k k
k
ES
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2 1
0 2
2
1
2 1
0
1
2 1
1
2 1
0
1
2 1
0
1
2 1
2 1
1
0
2 1
1
1
2 1
2 1
1
0
2 1
1
i
i
I S
k
k
K
k
k k
K E k P
dt
d
here

=
+
= =
(

; ;
:
1
2 1
0 2
max
|
|
.
|

\
|
+ +
(

=
I
M
K
I
S
K
P
dt
d
P
dt
d
1 1
max
Graphical Representation of Kinetic Data
Lineweaver-Burk Plot
Linearization experimental data of 1/(dP/dt) as function of S
-1
Eadie-Hofstee Plot
Linearization experimental data of (dP/dt) as function of (dP/dt) /S
Unexcessive emphasize points at low substrate concentration
The inhibition constant of K
i
can be determined from above plot result correlation
|
|
.
|

\
|
+ +
(

=
I
M
K
I
S
K
P
dt
d
P
dt
d
1 1
max
S
P
dt
d
K
P
dt
d S
P
dt
d
K
I
K
P
dt
d
P
dt
d
P
dt
d
P
dt
d
K
I
S
K
app
M
I
M
I
M
1 1 1
1
1 1
;
1
1 1
max max max max max

+
(

=
(

|
|
.
|

\
|
+
+
(

= =
(

|
|
.
|

\
|
+ +
S
P
dt
d
K
I
K P
dt
d
P
dt
d
P
dt
d
K
I
S
K
P
dt
d
I
M
I
M

|
|
.
|

\
|
+
(

=
(

=
|
|
.
|

\
|
|
|
.
|

\
|
+ + 1 ; 1 1
max max
I
K
K
K
K
I
K K
I
M
M
I
M
app
M
+ =
|
|
.
|

\
|
+ = 1
Inhibitory Mechanisms
ES S E
k
k

+
1
1
ESI I ES
i
i
k
k

+
E P ES
k
+

2
Derivatived Equation of Inhibitory Substrate Mechanism
| | | | ; 0 ,
0 0
= ESI ES | | | | ESI ES E E + + =
0
| | | |
| | | | | | | | | |
| | | | | |
| | | |
| | | | | | | |
| | | | | | ( ) ESI ES E S
k k
k
E S
k k
k
ES
I ES
k
k
k I ES k ES k ES k E S k
I ES
k
k
ESI
ESI k I ES k ESI
ESI k I ES k ES k ES k E S k ES
ESI ES
i
i
i i
i
i
i i dt
d
i i dt
d
dt
d
dt
d

+
=
+
=
=
|
|
.
|

\
|
+
=
=
+ =
= =


0
2 1
1
2 1
1
2 1 1
2 1 1
0
0 ; 0
| | | | | | | | ES I
k
k
E E ES I
k
k
E I ES
k
k
ES E E
i
i
i
i
i
i

|
|
.
|

\
|
+ =
|
|
.
|

\
|
+ + = + + =

1 , 1
0
Uncompetitive Inhibition
Haloenzyme
Inactive site
Active Site
Apoenzyme
substrate
inhibitor
cofactor
product
MECHANISM SCHEME
Inactive site
| |
| | | |
| | | |
| |
| |
S I
k
k
k k
k
E S
k k
k
ES
E S
k k
k
S I
k
k
k k
k
ES
E S
k k
k
ES I
k
k
S
k k
k
ES
ES I
k
k
E S
k k
k
ES
ES I
k
k
E E
i
i
i
i
i
i
i
i
i
i

|
|
.
|

\
|
+
+
+

+
=

+
=
)
`

|
|
.
|

\
|
+
+
+

+
=
|
|
.
|

\
|

|
|
.
|

\
|
+
+
+
|
|
.
|

\
|

|
|
.
|

\
|
+
+
=

|
|
.
|

\
|
+ =

1 1
1 1
1
1
1
2 1
1
0
2 1
1
0
2 1
1
2 1
1
0
2 1
1
2 1
1
0
2 1
1
0
| |
| |
| |
| |
| |
S
I
k
k
k
k k
I
k
k
S E
k
ES k P
dt
d
S
I
k
k
k
k k
I
k
k
S E
ES
S I
k
k
k
k k
S E
ES
S I
k
k
k
k k
S E
ES
S I
k
k
k k
k
S E
k k
k
ES
i
i
i
i
i
i
i
i
i
i
i
i
i
i
+
+
+
+

= =
+
+
+
+

=

|
|
.
|

\
|
+ +
+

=

|
|
.
|

\
|
+ +
+

=

|
|
.
|

\
|
+
+
+

+
=

1
1
1
1
1
1
1 1
1
2 1
0
2
2
1
2 1
0
1
2 1
0
1
2 1
0
2 1
1
0
2 1
1
I
k
k
K
S
S
I
k
k
P
dt
d
P
dt
d
i
i
M
i
i
+
+

+
(

1
1
max
:
1
;
1
:
; ;
:
max
max
1
2 1
0 2
max
then
I
k
k
K
K
I
k
k
P
dt
d
P
dt
d
if
k
k k
K E k P
dt
d
here
i
i
M app
M
i
i
app
S
+
=
+
(

=
(

+
= =
(

app
M
app
K S
S P
dt
d
P
dt
d
+

=
max
Graphical Representation of Kinetic Data
Lineweaver-Burk Plot
Linearization experimental data of 1/(dP/dt) as function of S
-1
S
P
dt
d
K
P
dt
d
P
dt
d
P
dt
d
S
P
dt
d
K S
app
app
M
app app
M
1 1 1
;
1 1
max max max

|
|
|
|
|
.
|

\
|
(

+
|
|
|
|
|
.
|

\
|
(

= =
(

+
app
M
app
K S
S P
dt
d
P
dt
d
+

=
max

Das könnte Ihnen auch gefallen