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Michaelis-Menten Parameters

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enzymes

It is important to have a feel for the magnitudes of

the kinetic constants, k and 1/K M, for certain

enzymes. The table in the next slide shows the

range of values encountered for several different

enzymes. Notice that almost all the experiments

reported were performed at moderate temperatures

and pH values. The exception is pepsin, which has

the task of hydrolyzing proteins in the acid

environment of the stomach. Consequently, the

enzyme has the greatest activity under the acidic

conditions employed in the experimental

determination of its kinetic parameters.

enzymes

Enzyme

Pepsin

Trypsin

Chymotryp

sin

Substrate

Temp,

C

pH

k3 , s 1

1/KM, M

1

31.6

4.0 0.00108

530

Carbobenzoxy-Lglutamyl-L-tyrosine

31.6

4.0 0.00141

560

Benzoyl-Largininamide

25.5

7.8

27.0

480

Chymotrypsinogen

19.6

7.5

2,900

<770

Saturin

24.5

7.5

13,100

400

Benzoyl-L-arginine

ester

25.0

8.0

26.7

12,500

Methyl

hydrocinnamate

25.0

7.8

0.026

256

Methyl dl--chloro-phenylpropionate

25.0

7.8

0.135

83.3

enzymes

Enzyme

Substrate

Methyl l-phenyllactate

Temp,

C

pH

25.0

7.8

k3 , s 1

1/KM, M

1

1.38

100

Evaluation of Michaelis-Menten

Parameters

A series of batch runs with different

levels of substrate concentration is

made in order to estimate the values

of the kinetic parameters. The results

are plotted graphically so that the

validity of the kinetic model can be

tested and the values of the kinetic

parameters can be estimated.

plot r against CS

KM is equal to CS when r = 0.5rmax.

However, this is an unsatisfactory

plot in estimating rmax and KM

because it is difficult to test the

validity of the kinetic model.

Therefore, the Michaelis-Menten

equation is usually rearranged so

that the results can be plotted as

a straight line.

rearranged to be expressed in

linear form. Some of the better

known methods are:

(a) Langmuir plot

(b) Lineweaver-Burk plot

(c) Eadie-Hofstee plot

LANGMUIR PLOT

Also known as Hanes-Woolf plot

Proponents of this method are:

Charles Samuel Hanes

Barnet Woolf

for the treatment of data from the

adsorption of gas on a solid surface.

CS K M

1

CS

r

rmax rmax

Refer to Figure 2.4, p. 24, James Lee

LINEWEAVER-BURK PLOT

Also known as double reciprocal

plot

Proponents are:

Hans Lineweaver

Dean Burk

KM 1

1

1

r rmax rmax C S

Refer to Figure 2.5, p. 25, James Lee

Slope: KM/rmax

Intercept: 1/rmax

EADIE-HOFSTEE DIAGRAM

Also known as:

Woolf-Eadie-Augustinsson-Hofstee

Eadie-Augustinsson

r rmax K M

r

CS

Slope: KM

Intercept: rmax

Observations:

The Lineweaver-Burk plot is more often

employed than the other two plots

because it shows the relationship between

the independent variable CS and the

dependent variable r. However, 1/r

approaches infinity as CS decreases, which

gives undue weight to inaccurate

measurements made at low substrate

concentrations, and insufficient weight to

the more accurate measurements at high

substrate concentrations. The points on

the line in Figure 2.5 represent seven

equally spaced substrate concentrations.

Observations

The Eadie-Hofstee plot gives slightly

better weighting of the data than the

Lineweaver-Burk plot. A disadvantage of

this plot is that the rate of reaction r

appears in both coordinates while it is

usually regarded as a dependent variable.

Based on the data distribution, the

Langmuir plot is the most satisfactory of

the three, since the points are equally

spaced.

Summary:

In conclusion, the values of MM kinetic

parameters, rmax and KM can be

estimated as follows:

1. Make a series of batch runs with

different levels of substrate

concentration at a constant initial

enzyme concentration and measure the

change of product or substrate with

respect to time.

2. Estimate the initial rate of reaction from

the CS or CP versus time curves for

different initial substrate concentrations.

Summary:

In conclusion, the values of MM kinetic

parameters, rmax and KM can be

estimated as follows:

plotting one of the three plots

explained in this section. It is

important to examine the data

points so that you may not include

the points which deviate

systematically from the kinetic

model.

Example 2.3

From a series of batch runs with a

constant enzyme concentration, the

following initial rate data were

obtained as a function of initial

substrate concentration.

CS

(mmol/

L)

10

15

20

Initial

rxn rate,

r

(mmol/L

min)

0.20

0.22

0.30

0.45

0.41

0.50

0.40

0.33

parameters by employing the Langmuir plot,

the Lineweaver-Burk plot, the Eadie-Hofstee

plot. In evaluating the kinetic parameters, do

not include data points which deviate

systematically from the Michaelis-Menten

model and explain the reason for deviation.

b. Which of the three methods employed gives

the best estimate of the kinetic parameters?

c. Repeat part (a) by using all data.

Problem 2.4

Eadie (1942) measured the initial

reaction rate of hydrolysis of

acetylcholine (substrate) by dog

serum (source of enzyme) and

obtained the following data:

Substrate

Concn

(mol/L)

0.0032

0.0049

0.0062

0.0080

0.0095

Initial Rxn

Rate

(mmol/Lmi

n)

0.111

0.148

0.143

0.166

0.200

Problem 2.14

Eadie (1942) measured the initial

reaction rate of hydrolysis of

acetylcholine (substrate) by dog

serum (source of enzyme) in the

absence and presence of prostigmine

(inhibitor), 1.510 7 mol/L and

obtained the following data:

Problem 2.14

CS mol/L

r, mol/Lmin

Absence of

Prostigmine

Presence of

Prostigmine

0.0032

0.111

0.059

0.0049

0.148

0.071

0.0062

0.143

0.091

0.0080

0.166

0.111

0.0095

0.200

0.125

parameters in the presence and absence of

prostigmine by employing:

i. Langmuir plot

ii. Lineweaver-Burk plot

iii. Eadie-Hofstee plot

Problem 2.14

Given:

CS mol/L

r, mol/Lmin

Absence of

Prostigmine

Presence of

Prostigmine

0.0032

0.111

0.059

0.0049

0.148

0.071

0.0062

0.143

0.091

0.0080

0.166

0.111

0.0095

0.200

0.125

Problem 2.14

Required:

a. MM kinetic parameters (KM and rmax)

by employing

a.1 Langmuir plot

a.2 Lineweaver-Burk plot

a.3 Eadie-Hofstee plot

b. Is prostigmine competitive or

noncompetitive inhibitor?

Problem 2.14

Solution:

a.1 Langmuir Plot

CS K M

1

CS

r

rmax rmax

rmax = 0.3018

rmax = 0.3346

KM = 5.772110 3 KM = 0.0164

R = 0.9401

R = 0.9021

Problem 2.14

0.08

f(x) = 2.99x + 0.05

0.07

0.06

0.05

w/o inhibition

Linear (w/o inhibition)

0.04

w/ inhibition

Linear (w/ inhibition)

0.03

0.02

0.01

0

0

0.01

0.01

0.01

0.01

0.01

0.01

Problem 2.14

Solution:

a.2 Lineweaver-Burk Plot

KM 1

1

1

r rmax rmax C S

rmax = 0.2752

rmax = 0.2613

KM = 4.730310 3 KM = 0.0115

R = 0.9563

R = 0.9783

Problem 2.14

18

f(x) = 0.04x + 3.83

16

14

12

10

w/o inhibitor

Linear (w/o inhibitor)

w/ inhibitor

Linear (w/ inhibitor)

0

50

100

150

200

250

300

350

Problem 2.14

Solution:

a.3 Eadie-Hofstee Plot

r rmax K M

r

CS

rmax = 0.2645

rmax = 0.2555

KM = 4.273110 3 KM = 0.0110

R = 0.8114

R = 0.8256

Problem 2.14

0.25

0.2

f(x) = - 0x + 0.26

0.15

w/o inhibitor

Linear (w/o inhibitor)

w/ inhibitor

f(x) = - 0.01x + 0.26

0.1

0.05

0

10

15

20

25

30

35

40

Problem 2.14

Solution:

Summary

Langmuir

Lineweaver-Burk

Eadie-Hofstee

rmax,

mol/Lmi

n

0.3018

0.3346

0.2752

0.2613

0.2645

0.2555

KM,

mol/L

5.7721

10 3

0.0164

4.7303

10 3

0.0115

4.2731

10 3

0.0110

0.9401

0.9021

0.9563

0.9783

0.8114

b. Since

maximum

reaction

rate

did 0.8256

not change greatly relative to KM, the

inhibitor is COMPETITIVE.

Problem 2.14

Solution:

Summary

Langmuir

MM

w/o

kinetic

prostigmi

paramete

ne

rs

Lineweaver-Burk

Eadie-Hofstee

w/

prostigmi

ne

w/o

prostigmi

ne

w/

prostigmi

ne

w/o

prostigmi

ne

w/

prostigmi

ne

rmax,

mol/Lmin

0.3018

0.3346

0.2752

0.2613

0.2645

0.2555

KM, mol/L

5.77211

03

0.0164

4.73031

03

0.0115

4.27311

03

0.0110

b. Since

maximum

reaction

rate 0.9783

did not change

0.9401

0.9021

0.9563

0.8114

greatly relative to KM, the inhibitor is

COMPETITIVE.

0.8256

Problem 2.17

The initial rate of reaction for the

enzymatic cleavage of

deoxyguanosine triphosphate was

measured as a function of initial

substrate concentration as follows

(Kornberg et al., J. Biol. Chem., 233,

CS (mol/L)

r (mol/Lmin)

159, 1958):

6.7

3.5

1.7

0.30

0.25

0.16

Problem 2.17

a. Calculate the Michaelis-Menten

constants of the above equation by:

i. Langmuir plot

ii. Lineweaver-Burk plot

iii. Eadie-Hofstee plot

Problem 2.17

b. When the inhibitor was added, the initial

reaction rate was decreased as follows:

CS

(mol/L)

Inhibitor

r

(mol/Lmi

n)

6.7

146

0.11

3.5

146

0.08

1.7

0.06

Is this competitive

or146

noncompetitive

inhibition? Justify your answer by showing

the effect of the inhibitor graphically

employing the three plots. [Contributed

by Professor Gary F. Bennett, The

University of Toledo, Toledo, OH]

Problem 2.17

Given:

CS (mol/L)

r (mol/Lmin)

6.7

3.5

1.7

0.30

0.25

0.16

CS

(mol/L)

Inhibitor

6.7

3.5

1.7

146

146

146

r

(mol/Lmi

n)

0.11

0.08

0.06

Problem 2.17

Required:

a. MM kinetic parameters (rmax and KM)

with and without the presence of the

inhibitor by employing:

i. Langmuir plot

ii. Lineweaver-Burk plot

iii. Eadie-Hofstee plot

b. Is the inhibition competitive or

noncompetitive?

Problem 2.17

Solution:

a.1 Langmuir Plot

CS K M

1

CS

r

rmax rmax

w/o inhibitor:

w/ inhibitor:

rmax = 0.4215

rmax = 0.1567

KM = 2.6317 KM = 2.9807

R = 0.9968

R = 0.9916

Problem 2.17

70

60

50

40

w/o inhibitor

Linear (w/o inhibitor)

w/ inhibitor

30

20

10

0

1

Problem 2.17

Solution:

a.2. Lineweaver-Burk plot

KM 1

1

1

r rmax rmax C S

w/o inhibitor:

w/ inhibitor:

rmax = 0.4511

rmax = 0.1415

KM = 3.0566 KM = 2.3613

R = 0.9961

R = 0.9876

Problem 2.17

18

16

14

12

10

w/o inhibitor

Linear (w/o inhibitor)

w/ inhibitor

f(x) = 6.78x + 2.22

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Problem 2.17

Solution:

a.3. Eadie-Hofstee

r rmax K M

r

CS

w/o inhibitor:

w/ inhibitor:

rmax = 0.4336

rmax = 0.1457

KM = 2.8096 KM = 2.5083

R = 0.9781

R = 0.9564

Problem 2.17

0.35

0.3

0.25

0.2

w/o inhibitor

Linear (w/o inhibitor)

w/ inhibitor

0.15

0.1

0.05

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

Problem 2.17

Solution:

Summary

Langmuir

Lineweaver-Burk

Eadie-Hofstee

MM

kinetic

paramete

rs

w/o

inhibitor

w/

inhibitor

w/o

inhibitor

w/

inhibitor

w/o

inhibitor

w/

inhibitor

rmax,

mol/Lmin

0.4215

0.1567

0.4511

0.1415

0.4336

0.1457

KM, mol/L

2.6317

2.9807

3.0566

2.3613

2.8096

2.5083

0.9968

0.9916

0.9961

0.9263

0.9781

0.9564

rmax, then the inhibition is NONCOMPETITIVE.

The enzyme, cathepsin, hydrolyzes Lglutamyl-L-tyrosine to carbobenzoxy-Lglutamic acid and L-tyrosine. It has been

found (Frantz and Stephenson, J. Biol.

Chem., 169, 359, 1947) that the glutamic

acid formed in the hydrolysis, inhibits

(competitively) the progress of the

reaction by forming a complex with

cathepsin. The course of the reaction is

followed by adding tyrosine decarboxylase

which evolves CO2.

Substrate, mol/mL

Inhibitor, mol/mL

Rate of CO2

Generation,

mol/mLmin

4.7

0.0434

4.7

7.57

0.0285

4.7

30.30

0.0133

10.8

0.0713

10.8

7.58

0.0512

10.8

30.30

0.0266

30.3

0.1111

30.3

7.58

0.0909

30.3

30.30

0.0581

Calculate (a) the value of MichaelisMenten constants of the enzyme, KS,

and (b) the dissociation constant of

enzyme-inhibitor complex, KI.

[Contributed by Professor Gary F.

Bennett, The University of Toledo,

Toledo, OH]

Given:

CS, mol/mL

CI, mol/mL

Rate of CO2

Generation, r,

mol/mLmin

4.7

0.0434

10.8

0.0713

30.3

0.1111

4.7

7.57

0.0285

10.8

7.58

0.0512

30.3

7.58

0.0909

4.7

30.30

0.0133

10.8

30.30

0.0266

30.3

30.30

0.0581

Required:

a. MM constant of the enzyme, KS

b. Dissociation constant of enzymeinhibitor complex, KI

Solution:

Any method can be used to solve for

KM and rmax

*By Langmuir Plot

CS K M

1

CS

r

rmax r max

@CI = 0

rmax = 0.1569 mol/mLmin R = 0.9997

KM = 12.5840 mol/mL

Solution:

@ CI = 7.58 mol/mL

rmax = 0.1537 mol/mLmin

0.9993

KM C

=I =

21.0719

@

30.30 mol/mL

rmax = 0.1560 mol/mLmin

0.9967

KM = 51.3369 mol/mL

R=

R=

Solution:

It is observed from the values of KM

and rmax that the inhibition is

CI

is given

K MI K S 1KMI

COMPETITIVE. Hence,

KI

by:

KMI = 21.0719 mol/mL @ CI = 7.58 mol/mL

KMI = 51.3369 mol/mL @ CI = 30.30 mol/mL

Solution:

Making use of the values solved for

KMI:

KMI = 21.0719 mol/mL @ CI = 7.58

mol/mL

We

now have two equations and two

KMIunknowns

= 51.3369

mol/mL

@ CI = 30.30

given

by:

7.58

21.0719 K S 1

KI

mol/mL

30.30

51.3369 K S 1

KI

Solution:

Solving the two equations

simultaneously. We arrive at:

KS = 10.9747 mol/mL

KI = 8.2387 mol/mL

Solution:

Any method can be used to solve for

KM and rmax

*By Lineweaver-Burk Plot

KM 1

1

1

r rmax r max C S

@CI = 0

rmax = 0.1516 mol/mLmin R = 0.9996

KM = 11.7725 mol/mL

Solution:

@ CI = 7.58 mol/mL

rmax = 0.1463 mol/mLmin

0.9996

KM C

=I =

19.5076

@

30.30 mol/mL

rmax = 0.1406 mol/mLmin

0.9997

KM = 45.1406 mol/mL

R=

R=

Solution:

It is observed from the values of KM

and rmax that the inhibition is

CI

given by:

K MI K S 1K

MI is

COMPETITIVE. Hence,

KI

KMI = 19.5076 mol/mL @ CI = 7.58 mol/mL

KMI = 45.1406 mol/mL @ CI = 30.30 mol/mL

Solution:

Making use of the values solved for KMI:

KMI = 19.5076 mol/mL @ CI = 7.58

mol/mL

KMI =

45.1406

mol/mL

@ CI and

= 30.30

We

now

have two

equations

two

unknowns

by:

7.58

mol/mL 19given

.5076 K 1

S

K I

30.30

45.1406 K S 1

KI

Solution:

Solving the two equations

simultaneously. We arrive at:

KS = 10.9557 mol/mL

KI = 9.7107 mol/mL

Solution:

Any method can be used to solve for

KM and rmax

*By Eadie-Hofstee Plot

r rmax K M

r

CS

@CI = 0

rmax = 0.1545 mol/mLmin R = 0.9976

KM = 12.1869 mol/mL

Solution:

@ CI = 7.58 mol/mL

rmax = 0.1512 mol/mLmin

0.9970

KM C

=I =

20.4919

@

30.30 mol/mL

rmax = 0.1523 mol/mLmin

0.9931

KM = 49.7729 mol/mL

R=

R=

Solution:

It is observed from the values of KM

and rmax that the inhibition is

KMI

C I is given

COMPETITIVE. Hence,

K MI K S 1

KI

by:

Making use of the values solved for KMI:

KMI = 20.4919 mol/mL @ CI = 7.58 mol/mL

KMI = 49.7729 mol/mL @ CI = 30.30 mol/mL

Solution:

Making use of the values solved for

KMI:

KMI = 20.4919 mol/mL @ CI = 7.58

mol/mL

We

now have two equations and two

KMIunknowns

= 49.7729

mol/mL

@ CI = 30.30

given

by:

7.58

20.4919 K S 1

KI

mol/mL

30.30

49.7729 K S 1

KI

Solution:

Solving the two equations

simultaneously. We arrive at:

KS = 10.7230 mol/mL

KI = 8.3203 mol/mL

Bioreactor device within which biochemical

transformations are caused by the action of

enzymes or living cells

Fermenter bioreactor in which the

transformation is carried out by living cells or

in vivo cellular components (that is, enzymes)

Enzyme reactor bioreactor employing

enzymes; the term is used to distinguish it

from the bioreactor in which employs living

cells

The simplest reactor configuration for any

enzyme reaction is the batch mode. A

batch enzyme reactor is normally

equipped with an agitator to mix the

reactant, and the pH of the reactant is

maintained by employing 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.

MM General Rate Equation

dCS

rmaxC S

dt

K M CS

Rearranging, and integrating

yields

CS

t

K M CS

dCS rmaxdt

CS

CS 0

Integrated Form:

CS0

C S0 C S rmaxt

CS

The eqn above shows how CS is

changing with respect to time. With

known values of rmax and KM, the

change in CS with time in a batch

K M ln

reactor)

In a plug-flow enzyme reactor, the substrate

enters one end of a cylindrical tube which is

packed with immobilized enzyme and the

product stream leaves at the other end. The

long tube and lack of stirring device prevents

complete mixing of the fluid in the tube.

Therefore, the properties of the flowing

stream will vary in both longitudinal and

radial directions. Since the variation in radial

direction is small compared to that in the

longitudinal direction, it is called a plug-flow

reactor. If a plug-flow reactor is operated at

steady-state, the properties will be constant

Reactor

C S 0 C S

ln

C S0

C

S

K M rmax

t

C S0

ln

C

S

A continuous stirred-tank

reactor (CSTR) is an ideal

reactor w/c is based on

the assumption that the

reactor contents are well

mixed.

Therefore, the concentrations of the various

components of the outlet stream are

assumed to be the same as the

concentrations of these components in the

reactor.

Substrate balance in CSTR:

Input Output + Generation =

Accumulation

dCS

FC S0 FC S rSV V

dt

where:

F = flow rate

V = volume

rS = rate of substrate consumption

dCS/dt = change of substrate concn

For a batch reactor: F = 0 and rS = dCS/dt

For a steady-state CSTR, dCS/dt = 0, and

rS is given by Michaelis-Menten rate

equation

rmaxC S

F as: 1

D

V

C S0 C S K M C S

where:

D = dilution ratio

Rearranging the equation above gives the

linear relationship

CS K M

C S

rmax

C S0 C S

Problem 2.6

A carbohydrate (S) decomposes in the presence

of an enzyme (E). The Michaelis-Menten

kinetic parameters were found as follows:

KM = 200 mol/m3

rmax = 100 mol/m3min

a. Prepare a CS versus t curve when the initial

substrate concentration is 300 mol/m3.

curve you calculated in part (a)

experimentally. Estimate KM and rmax by

plotting the (CS0 CS)/ln(CS0/CS) versus

t/ln(CS0/CS) curve.

Problem 2.6

c. Chemostat (continuously stirred-tank reactor)

runs with various flow rates were carried out.

If the inlet substrate concentration is 300

mol/m3 and the flow rate is 100 cm3/min,

what is the steady-state substrate

concentration of the outlet? The reactor

volume is 300 cm3. Assume that the enzyme

concentration in the reactor is constant so

that the same kinetic parameters can be

used.

Problem 2.6

Given:

KM = 200 mol/m3

rmax = 100

mol/m3min

CS0 = 300 mol/m3

Problem 2.6

Required:

a. CS vs t graph

b. KM and rmax suppose values

obtained in (a) were determined

experimentally.

c. CSTR configuration, find CS outlet

CS0 = 300 mol/m3

F = 100 cm3/min

V = 300 cm3

Problem 2.6

Solution

a. Table

K M ln

C S0

CS

C S

C S rmaxt

CS, mol/m3

t, min

250

0.8646

200

1.8109

150

2.8863

100

4.1972

50

6.0835

---------

Problem 2.6

Solution

b. Table for Linear Graph:

C S0 C S

C S0

ln

K M rmax

t

C S0

C

S

CS, mol/m3

ln

C

S

t, min

By LR:

KM = -200.0639 mol/m3

rmax = 100.0165

t/ln(CS0/CS)

(CS0

mol/m3min

CS)/ln(CS0/CS)

250

0.8646

4.7422

274.2407

200

1.8109

4.4662

246.6303

150

2.8863

4.1641

216.4043

100

4.1972

3.8205

182.0478

50

6.0835

3.3953

139.5277

----------

----------

----------

Problem 2.6

Solution

c. Continuous stirred tank reactor (CSTR)

By applying the derived equation:

CS K M

rmaxC S

C S0 C S

CS = 164.5751 mol/m3

Other Root = -364.6895

Problem 2.7

The KM value of an enzyme is known

to be 0.01 mol/L. To measure the

maximum reaction rate catalyzed by

the enzyme, you measured the initial

rate of reaction and found that 10

percent of the initial substrate

concentration was consumed in 5

minutes. The initial substrate

concentration is 3.410 4 mol/L.

Assume that the reaction can be

Problem 2.7

a. What is the maximum reaction rate?

b. What is the concentration of

substrate after 15 minutes

CS = 2.476210 4 mol/L

Problem 2.8

A substrate is converted to a product by the

catalytic action of an enzyme. Assume the

Michaelis-Menten kinetic parameters for this

enzyme reaction are:

KM = 0.03 mol/L

rmax = 13 mol/Lmin

a. What should be the size of a steady-state CSTR

to convert 95 percent of the incoming

substrate (CS0 = 10 mol/L) with a flow rate of

10 L/h.

b. What should be the size of the reactor if you

employ a plug-flow reactor instead of the CSTR

in (a)?

Problem 2.8

Given

F = 10 L/h

CS0 = 10

mol/L

KM = 0.03 mol/L

rmax = 13

mol/Lmin

CS = ?

95% conversion

Problem 2.8

Required

a. V for 95% conversion

b. V if plug-flow reactor is employed

Problem 2.9

A substrate is decomposed in the presence of an

enzyme according to the Michaelis-Menten

equation with the following kinetic parameters:

KM = 10 g/L

rmax = 7 g/Lmin

If we operate two one-liter CSTRs in series at

steady-state, what will be the concentration of

substrate leaving the second reactor? The flow

rate is 0.5 L/min. The inlet substrate

concentration is 50 g/L and the enzyme

concentration in the two reactors is maintained

at the same value all of the time. Is the tworeactor system more efficient than one reactor

Problem 2.1

In order to measure the enzyme activity and

the initial rate of reaction, 5 mL of cellobiose

(100 mol/mL) and 44 mL of buffer solution

were placed in a stirred vessel. The reaction

was initiated by adding 1 mL of enzyme (glucosidase) solution which contained 0.1 mg

of protein per mL. At 1, 5, 10, 15, and 30

minutes, 0.1 mL of sample was removed from

reaction mixture and its glucose content was

measured. The result were as follows:

Problem 2.1

Time, min

Glucose Concentration,

mol/mL)

0.05

0.23

10

0.38

15

0.52

30

1.03

units/mL of enzyme solution and in units/mg

protein? A unit is defined as the enzyme activity

which can produce 1 mol of product per minute?

b. What is the initial rate of reaction?

Activity

Some chemical and physical conditions

affect the rate of an enzyme

reaction. Some of these factors are

the concentration of various

components (substrate, product,

enzyme, cofactor, and so on), pH,

temperature, and shear.

Effect of pH

The pH of the solution strongly

influences the rate of enzyme

reaction both in vivo and in vitro. The

optimum pH is different for each

enzyme.

Examples:

Pepsin (from stomach) 2 < pH < 3.3

Amylase (from saliva) pH = 6.8

Chymotripsin (from pancreas) 7 < pH

<8

Effect of pH

pH shows a bell-shaped curve.

Effect of pH

Reasons that the rate of enzyme

reaction is influenced by pH can be

explained as follows:

1. Enzyme is a protein which consists

of amino acid residues

Effect of pH

2. The amino acid residues possess

basic, neutral, or acid side groups

which can be positively or

negatively charged at any given pH.

pH is increased, glutamic acid is ionized.

Effect of pH

Ionization is expressed according to:

In eqbm,

When C ,CpH

is equal to pK. For

A

A

glutamic acid, pK = 4.5.

Effect of pH

Lysine is basic in the range of higher

pH value. As the pH is decreased,

lysine is ionized as

of the residues are ionized.

Effect of pH

3. An enzyme is catalytically active

when the amino acid residues at the

active site each possess a particular

charge. Therefore, the fraction of the

catalytically active enzyme depends

on the pH.

Effect of pH

Conclusion

Suppose that one residue of each of these

two amino acids, glutamic acid and lysine,

is present at the active site of an enzyme

molecule and that, for example, the

charged form of both amino acids must be

present if that enzyme is to function. Since

glutamic acid is charged when its pH 4.5

and lysine is charged when its pH 10.0,

the enzyme will be most active when 4.5

pH 10.0 as shown in the given figure.

Effect of Temperature

The rate of enzyme-catalyzed

reactions increases with temperature

up to a certain limit. Above a certain

temperature, enzyme activity

decreases with temperature because

of enzyme denaturation.

Effect of Temperature

Figure below depicts the variation of

reaction rate with temperature and

the presence of an optimal

temperature.

Effect of Temperature

Temperature activation: ascending

part; the rate varies according to the

Arrhenius equation in this region.

r k 2C E

where

k 2 Ae

Ea / RT

Effect of Temperature

where

Ea = activation energy

CE = active enzyme concentration

Plot of ln(r) versus 1/T results in a line

with slope Ea/R

Effect of Temperature

Temperature deactivation/thermal

denaturation

dCE

kd CE

dt

C E C E0 e kd t

temperature according to the

Ea / RT

Arrhenius eqn k d Ad e

Effect of Temperature

Consequently

r Ae Ea / RT C E0 e kd t

Effect of Shear

Enzymes had been believed to be

susceptible to mechanical force, which

disturbs the elaborate shape of an enzyme

molecule to such a degree that

denaturation occurs. The mechanical force

that an enzyme solution normally

encounters is fluid shear, generated

either by flowing fluid, the shaking of a

vessel, or stirring with an agitator. The

effect of shear on the stability of an

enzyme is important for the consideration

of enzyme reactor design, because the

Effect of Shear

Charm and Wong (1970) showed that the

enzymes catalase, rennet, and

carboxypeptidase were partially

inactivated when subjected to shear in a

coaxial cylinder viscometer. The remaining

activity could be correlated with a

dimensionless group . In the case of

catalase, about 50 percent of the activity

was lost when was 0.5107.

= shear rate

= time of exposure to shear

Effect of Shear

Thomas and Dunnill (1979) studied the

effect of shear on catalase and urease

activities by using a coaxial cylindrical

viscometer that was sealed to prevent any

air-liquid contact. They found that there

was no significant loss of enzyme activity

due to shear force alone at shear rates up

to 106 sec1. They reasoned that the

deactivation observed by Charm and Wong

(1970) was the result of combination of

shear, air-liquid interface, and some other

effects which are not fully understood.

Effect of Shear

Recently, this as further confirmed, as

cellulase deactivation due to the

interfacial effect combined with the shear

effect was found to be far more severe and

extensive than that due to the shear effect

alone (Jones and Lee, 1988).

1. T/F? The Ea calculated from the Arrhenius

equation gives an exact value.

Ans.: F. Ea is an average or apparent

value.

2. Describe the relationship between

temperature and kd and give examples.

Ans.: As the temperature increases, the rate

constant decreases when the equation is

plotted. The same is true when the

temperature decreases, the rate constant

increases. From this connection, the rate

3. Using the following information:

A = 11014 sec 1

Ea = 75103 J/mol

R = 8.314 J/molK

Calculate k at 27C with proper units.

4. Using the information in problem 3,

calculate k at 37C with proper units.

5. Calculate the value of Ea given:

k1 = 7.78107 at T1 = 273 K

k2 = 3.46105 at T2 = 298 K

Answers

kd = 8.8592/sec

kd =

23.3476/sec

Ea = 102,670.8716

J/mol

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