Académique Documents
Professionnel Documents
Culture Documents
Outline
Enzyme Kinetics
- Models for More Complex Enzyme Kinetics
- Effects of pH and Temperature
- Insoluble Substrates
Immobilized Enzyme Systems:
- Methods of Immobilization
- Diffusional Limitations in Immobilized
Enzyme Systems
Vm S
d S
v
"
n
dt
K m S
n
Allosteric Enzymes
The cooperativity coefficient can be determined by plotting ln
[v/(Vm-v)] versus ln[S] as
v
ln
n ln S ln K m"
Vm v
E S
ES
EP
k 1
KI
I
EI
Chung Yuan Christian University
Mapa Institute of Technology
ES
EI
K
,
KI
ES
EI
E 0 E ES EI and v k2 ES
'
m
Vm S
Vm S
v '
or v '
K m 1 I / K I S
K m , app S
I>0
Intercept
1/ Km
Chung Yuan Christian University
Mapa Institute of Technology
1 / K m , app
I=0
1
Vm
1
[S ]
E S
ES
EP
K m'
I
I
KI
EI S
ESI
K'
m
ES EI S
EI ESI
K
, KI
ES ESI
EI ESI
E 0 E ES EI ESI and v k2 ES
'
m
Vm
v
I K m'
1
1
S
K I
Chung Yuan Christian University
Mapa Institute of Technology
or v
Vm, app
K m'
1
I>0
I=0
1 / Vm , app
1 / Vm
1/ Km
1
[S ]
k2
E S
EP
ES
KI
I
ESI
ES
ESI
K
,
KI
ES
ESI
E 0 E ES ESI and
'
m
v k2 ES
Thermodynamics Research Laboratory
School of Chemical Engineering and Chemistry
Vm S
I
1
KI
v
K m'
S
I
1
KI
or
Vm , app S
K m' , app S
I>0
I=0
1 / Vm , app
1 / Vm
1 / K m , app 1 / K m
Chung Yuan Christian University
Mapa Institute of Technology
1
[S ]
Substrate
Inhibition
No
Inhibition
[S ]
S m ax
k2
E S
ES
EP
S
ES2
KS1
K S1
SES
,
ES 2
'
m
SE
ES
Vm S
v '
2
K m S S / K S1
1 / Vm
1/ Km
1
[S ]
Vm
v
K m'
1 S
or
1 K m' 1
1
v Vm S Vm
Vm
S
1
K S1
or
1
S
1
v K S1Vm Vm
Smax
'
m
K K S1
pH effects
Certain enzymes have ionic groups on their active sites, and
these ionic groups must be in a suitable form (acid or base) to
function.
Variations in the pH of the medium result in changes in the
ionic form of the active site and changes in the activity of the
enzyme and hence the reaction rate.
Changes in pH may also alter the three-dimensional shape of
the enzyme.
For these reasons, enzymes are only active over a certain pH
range.
The pH of the medium may affect the maximum reaction rate,
Km, and the stability of the enzyme; in some cases, the substrate
may contain ionic groups, and the pH of the medium affects the
affinity of the substrate to the enzyme.
Chung Yuan Christian University
Mapa Institute of Technology
E H
K2
K m'
k2
EH S
EHS
EH P
K1
EH 2
EH
S
EH
H
E
H
'
Km
, K1
, K2
EH 2
EHS
EH
E 0 E EH EH 2 EHS , v k2 EHS
Vm S
v
K
H
'
2
K m 1
S
K1
H
or
Vm S
v '
K m, app S
k2
SH E
ESH E HP
K1
S H
Vm S
v
K1
'
K m 1 S
H
100
0
10
Temperature, oC
60
Thermodynamics Research Laboratory
School of Chemical Engineering and Chemistry
v k 2 E
k 2 Ae Ea / RT
Where Ea is the activation energy and [E] is the active enzyme concentration.
d E
kd E
dt
or
E E0 ek t
d
Where [E0] is the initial enzyme concentration and kd is the denaturation constant.
k d Ad e Ed / RT
Where Ed is the deactivation energy.
Consequently,
v Ae Ea / RT E 0 e k d t
Chung Yuan Christian University
Mapa Institute of Technology
Vmax,S E
K eq E
K eq k des / k ads
The above equation assumes slow binding of enzyme (i.e., [E] ~ [E0]),
S0 is the number of substrate bonds available initially for breakage,
and kdes and kads refer to rates of enzyme desorption and adsorption
onto the insoluble matrix, respectively.
Chung Yuan Christian University
Mapa Institute of Technology
Entrapment
The physical enclosure of enzymes in a small space.
Matrix entrapment and membrane entrapment, including micro
encapsulation, are the two major methods of entrapment.
- matrices used for enzyme immobilization are usually
polymeric materials (e.g., Ca-alginate, agar, -carrageenin,
polyacrylamide, and collagen).
- however, some solid matrices (e.g., activated carbon, porous
ceramic, and diatomaceous earth) can also be used for this
purpose.
Chung Yuan Christian University
Mapa Institute of Technology
Assumptions:
- enzymes are bound and evenly
distributed on the surface of a
nonporous support material
Sb
- all enzyme molecules are
Bulk Substrate
equally active
Concentration
- substrate diffuses through a thin
liquid film surrounding the
support surface to reach the
Substrate
reactive surfaces
concentration
- the process of immobilization
profile in a
liquid film
has not altered the protein
around
structure
adsorbed
- the intrinsic kinetic parameters
enzymes.
(Vm, Km) are unaltered.
SS = Substrate
Concentration at
Surface
Nonporous
solid
support
Film thickness,
Chung Yuan Christian University
Mapa Institute of Technology
Vm' SS
J S k L Sb SS
K m SS
J or V
(A)
(B)
(B)
SS
Chung Yuan Christian University
Mapa Institute of Technology
v k L Sb ,
(for Da 1)
When the system is reaction limited (Da << 1), the reaction rate
is often expressed as
Vm' Sb
v
K m,app Sb
Vm'
Where: K m,app K m 1
k
S
K
L
b
m
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
SS
Sr2
r=0
Chung Yuan Christian University
Mapa Institute of Technology
r=R
Thermodynamics Research Laboratory
School of Chemical Engineering and Chemistry
d 2 S 2 d S
Vm" S
De 2
r dr K m S
dr
With boundary conditions:
[S] = [SS] at r = R and
d[S]/dr = 0 at r = 0.
Where Vm is the maximum reaction
rate per unit volume of support, and
De is the effective diffusivity of
substrate within the porous matrix.
S
S
,
SS
r
r ,
R
2 d S R 2Vm" S
or
S
D
r
d
r
S
dr
S e
d2S
Km
SS
d2S
2 dS
S
2
2
r dr
1 (S / )
dr
Vm" / K m
Where R
Thiele modulus
De
Chung Yuan Christian University
Mapa Institute of Technology
or
r d r S S De S
dr
d2S
d2S
2 dS
S
2
2
r dr
1 (S / )
dr
d S
rS N S 4R De
dr r R
2
Vm" SS
rS
K m SS
EFFECTIVENESS FACTOR,
0.01
0.1
1
0.4
= 10
0.2
10
20
40
100
THIELE MODULUS,
Theoretical relationship between the effectiveness factor and first-order
Thiele modulus for a spherical porous immobilized particle for various
values of , where is the dimensionless Michaelis constant.
Chung Yuan Christian University
Mapa Institute of Technology
3 1
1
tanh
Topic Assignments
Group 4: Bioenergetics; Glucose Metabolism
Group 5: Respiration; Aerobic Glucose Metabolism
Group 6: Role of ATPs; Role of coenzymes FAD and
NAD; Electron transport chain; Role of
mitochondria; How ATPs are produced in the
electron transport chain
Group 1: Metabolism of Nitrogenous Compounds;
Nitrogen Fixation;
Group 2: Metabolism of Hydrocarbons; Overview of
Biosynthesis;
Group 3: Overview of Anaerobic Metabolism;
Overview of Autotrophic Metabolism
Chung Yuan Christian University
Mapa Institute of Technology
END
Chung Yuan Christian University
Mapa Institute of Technology