Lecture 4

The Kinetics of Enzyme-Catalyzed

Reactions
 Effect of pH and temperature

-Enzyme are active only over small range of pH due to

the active site functional group charges (ionic form) and
the three dimensional shape of enzyme are pH-
dependent
-Certain enzyme have ionic group on their active sites,
and these ionic group must be in a suitable form (acid or
base) to function.
-Variation in pH of medium result in changes of:
-Ionic form of the active site
-Activity of enzyme, hence the reaction rate
-Affect the maximum reaction rate, Km
-stability of enzyme
 -Scheme to describe pH dependence of the enzymatic
reaction rate for ionizing enzymes.
-

Variation of enzyme activity

with pH for 2 different
enzymes

-Some cases, the substrate may contain ionic groups,

and the pH of medium affects the affinity of substrate to
enzyme.
-Refer to Eq. 3.44& Eq. 3.45
 ascending

Thermal
descending
Denaturation
occurred

The rate varies Variation of reaction rate with temperature

according to
Arrhenius equation
Restriction of enzyme mobility
in a fixed space = enzyme
immobilization
Allowing small MW
compound access to Retain high MW
enzyme compound
Prior to
Functional groups on support material are usually
activated by using chemical reagent such as
cyanogen bromide, carbodiimide and
glutaraldehyde
 Chemical reagent
Support
materials
with
functional
group
 Cross-linking of enzyme molecule

The agent used are: ____________,

____________, ________________

Cross-linking can be achieved in several ways:

1)
2)
3)

The disadvantages of cross-linking:

1)
2)
 Quiz

This is the schematic diagram of enzyme

immobilization methods:
Suggest the method of (a), (b), (c) and (d)?
 Diffusional Limitation in Immobilized
Enzyme System

Immobilized enzyme system normally includes

- insoluble immobilized enzyme
- soluble substrate, or product

They are heterogeneous systems

Substrate
HIGH
external diffusion

Immobilized
Sb
Enzyme

Low S concentration

DIFFUSION
DRIVING FORCE
external diffusion HIGH

Immobilized
Sb
Enzyme

REACTION

DIFFUSION
PRODUCT DRIVING FORCE
 HIGH

Immobilized
Sb
Enzyme

DIFFUSION
DRIVING FORCE
 HIGH

Immobilized
Sb
Enzyme
REACTION

PRODUCT
 Diffusional Limitation in
Immobilized Enzyme Systems
In immobilized enzyme systems,
the overall production rate is determined
by
- liquid film mass transfer (external diffusion)
substrate, product
- intraparticle mass transfer (internal diffusion)
substrate, product in porous supports
- enzyme catalysis reaction
Mass transfer coefficient
(cm/s)

Substrate
conc.
(g/cm3)
 Diffusional Limitation in
Immobilized Enzyme System
Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials
Ss
Sb
k2
E+S ES P E

Assume the enzyme catalyzed

reaction rate follows Michaelis-Menten
type kinetics.
Enzyme

Liquid Film Thickness, L

Ss: substrate concentration at the surface;

Sb: substrate concentration in bulk solution.
 Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials

Assume: Ss
Sb
-Enzyme are evenly distributed on the
surface of a nonporous support
material.
-All enzyme molecules are equally
active.
Enzyme
-Substrate diffuses through a thin
liquid film surrounding the support Liquid Film Thickness, L
No intraparticle diffusion
surface to reach the reactive surface.
-The process of immobilization has not altered the enzyme
structure and the intrinsic parameters (Vm, Km) are unaltered.
Enzyme
are bound
on surface

At steady state, the reaction

rate=mass transfer rate
Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials

The external diffusion rate Js (g/cm2-s):

J s k L ([Sb ] [S s ])
kL is the liquid mass transfer coefficient (cm/s).
If the product formation rate is :
Vm '[ S s ]
v
'

K m [S s ]
Vm ' the maximum reaction rate per unit surface area.
(g/cm2-s)
Graphical solution for reaction rate per unit of surface area
for enzyme immobilized on a non-porous support
Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials

When the system is strongly external diffusion

(liquid film mass-transfer) limited, [Ss]0,
the overall reaction rate is equal to the rate:

v k L [ Sb ] Da>>1

The system behaves as pseudo first order.

The rate is a linear function of bulk substrate concentration.

Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials
When the system is strongly reaction limited,
[Sb] [Ss]
the overall reaction rate is equal to the rate:

Vm '[ Sb ]
v Da << 1
K m, app [ Sb ]

where
Vm'
K m,app K m 1
k L ([ Sb ] K m )
Km,app is increased. It is a function of mixing speed and Sb.
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
- Substrate diffuses through the tortuous
pathway within the porous support to reach
the enzyme.
- Substrate reacts with enzyme on the pore
surface.

Ex. Spherical support particles

Sr
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix

Assume:
- Enzyme is uniformly distributed in a
spherical support particle.
- The reaction kinetics follows Michaelis-
Menten kinetics.
- There is no external diffusion limitation,
(no partitioning of the substrate between
exterior and interior of support).
A steady state: Diffusion rate =reaction rate
 reaction rate with intraparticle diffusion limitation

reaction rate withoutdiffusion limitation.

Relationship of effectiveness factor with the size of
immobilized enzyme particle and enzyme loading