By:

Engr. Vera Marie L. Lanaria

ChE Department
CIT University
Kinetics of Enzyme
Reactions
deals with the rate of enzyme reaction and
how it is affected by various chemical and
physical conditions
it provides information about the basic
mechanism of the enzyme reaction and
other parameters that characterize the
properties of the enzyme
rate equations can be applied in calculating
reaction time, yields, & optimum economic
conditions needed in designing bioreactors
Let S be the substrate (reactant)
E be the enzyme
P be the product
A simple reaction would be:
S + E P

Rate of reaction can be expressed in terms
of: r = v
s
= - dS/dt
or: v
p
= dP/dt
Victor Henri (1902, a French physical
chemist) proposed a quantitative theory of
enzyme kinetics and formulated the rate
equation:
v = v
max
S
K
M
+ S
In 1913, Leonor Michaelis (German bio-
chemist) and Maud Menten (Canadian
physician) continued the work of Henri in
which later on it becomes the Michaelis-
Menten model


(Emil Fischer 1894)
(Daniel Koshland 1958)
Assumptions:
The total enzyme concentration stays
constant during reaction, that is,
C
Eo
= C
ES
+ C
E

The amount of an enzyme is very small
compared to the amount of substrate; so
the formation of enzyme-substrate complex
does not significantly deplete the substrate.
The product concentration is so low that
product inhibition may be considered
negligible.
Langmuir plot (or Hanes Woolf plot)
Lineweaver-Burk plot
Eadie-Hofstee plot
Sample Problem:
From a series of batch runs with a constant
enzyme concentrations, the following initial
rate data were obtained as a function of
initial substrate concentration. (Refer to the
next slide for the data.) Evaluate the
Michaelis-Menten kinetic parameters by
employing the 3 linear forms or plots. In
evaluating the parameters do not include
data points which deviate systematically
from the Michaelis-Menten model.


S (mmol/L) - v (mmo/L-min)
1 - 0.20
2 - 0.22
3 - 0.30
5 - 0.45
7 - 0.41
10 - 0.50
15 - 0.40
20 - 0.33
Solution:
Examination of the data reveals that as the
substrate concentration (S) increased up to
10 mmo/L, the rate increased. However, the
further increases in the S to 15 mmol/L, the
initial reaction rate decreased. This behavior
may be due to substrate or product inhibition.
Since the Michaelis-Menten equation does
not incorporate the inhibition effects, thus
these two data points will be included.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0 5 10 15 20 25
S (mmo/L)
v

(
m
m
o
l
/
L
m
i
n
)
Langmuir Plot
y = 1.5866x + 4.6417
R
2
= 0.9497
0
5
10
15
20
25
0 2 4 6 8 10 12
S (mmol/L
S
/
v

(
m
i
n
)
From the line equation:
y = 1.5866x + 4.6417

slope = 1/v
max
= 1.5866
v
max
= 1/1.5866
v
max
= 0.63 min
-1

y-intercept = K
M
/v
max
= 4.6417
K
M
= (4.6417)(0.63)
K
M
= 2.92 mmol/Lmin
2

Lineweaver-Burk Plot
y = 3.4575x + 1.945
R
2
= 0.8463
0
2
4
6
0 0.2 0.4 0.6 0.8 1 1.2
1/S
1
/
v
From the line equation:
y = 3.4575x + 1.945

y-intercept = 1/v
max
= 1.945
v
max
= 1/1.945
v
max
= 0.514 min
-1


slope = K
M
/v
max
= 3.4575
K
M
= 3.4575(0.514)
K
M
= 1.78 mmol/Lmin
2
Eadie-Hofstee Plot
y = -1.8923x + 0.5386
R
2
= 0.6618
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0 0.05 0.1 0.15 0.2 0.25
v/S
V
From the line equation:
y = -1.8923x + 0.5386

y-intercept = v
max
= 0.5386
v
max
0.54 min
-1


slope = -K
M
= -1.8923
K
M
= 1.8923
K
M
1.89 mmol/Lmin
2
Bioreactor is a device/equipment within
which biochemical transformation are
caused by the action of enzyme or living
cells
Classifications of bioreactor:
1) Batch
2) Steady-State Plug-Flow Reactor (PFR)
3) Continuous Stirred-Tank Reactor (CSTR)
is normally equipped with agitator
pH is maintained by using either a buffer
solution or a pH controller
an ideal batch reactor is assumed to be well
mixed so that the contents are uniform in
composition at all times
Reaction Mechanism:
- dS = v
max
S
dt K
M
+ S
rearranging & integrating:
-(K
M
+S).dS/S = v
max
.dt
passing the limits: at t=0 ; S = S
o

at t=t ; S = S
- K
M
ln(S/S
o
) (S S
o
) = v
max
t
K
M
ln(S
o
/S) + (S
o
S) = v
max
t

the substrate enters one end of a cylindrical
tube which is packed with immobilized
enzyme and the product stream leaves at
the other end
properties of flowing stream will vary in both
longitudinal and radial directions since there
is no agitator used

since the variation in the radial direction is
small compared to that in the longitudinal
direction, its called plug-flow reactor
if PFR is operated at steady-state, the
properties will be constant with respect to
time
equation in batch reactor can be applied to
an ideal steady-state PFR, however, the
time, t, should be replaced with the
residence time,
S
o
S = -K
M
+ v
max
.
ln(S
o
/S) ln(S
o
/S)
is an ideal reactor which is based on the
assumption that the reactor contents are
well mixed
continuous operation can increase the
productivity significantly by eliminating the
downtime
easy to automate
substrate balance can be set up as follows:
Input - Output + Generation = Acc.
F(S
o
) - F(S) + r
s
V = V(dS/dt)
where: F = flow rate
V = volume of the reactor
r
s
= rate of substrate consumption
but for steady-state CSTR, the concentration
of substrate should be constant, thus
dS/dt = 0
and if Michaelis-Menten equation can be
used for the rate of substrate consumption,
then the equation can be arranged as:
F = D = 1/ = v
max
S .
V (S
o
S)(K
M
+ S)
where: D = is known as dilution rate
(Note: Its common in biochemical reaction to
use the term dilution rate, than the term
residence time.)
S = -K
M
+ (v
max
S)(S
o
S)
Inhibitor can decrease the rate of reaction
either competitively, non-competitively,
partially competitively, or mixed
Other Factors that influences
Enzyme Activity
temperature
pH
effect of shear