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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, it’s 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: It’s 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

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