ENZYME KINETICS

V
Kelompok Bioproses Kelas C :
1. Gapenda Sari Kaban (13-066)
2. Evan Kurniawan (13-067)
3. Vivian (13-073)
4. Yudi Faradey P (13-081)
5. Alexander (13-089)
6. Andre Prasetia W (13-090)
7. Trecy Kartika (13-093)
8. Maria Paula S (13-094)

ENZYME ASSAY
The analysis for enzyme activity.
The results obtained from enzyme assays are not in weight of enzyme, but units of
catalytic activity.
One unit of enzyme activity
= the amount of enzyme which will catalyze the formation of 1 mol
product per minute under specified conditions
Enzyme assays must be carried out under conditions that are optimal for activity,
with the pH, temperature and concentration of reagents used in a predetermined
manner.
Enzyme assays must be made under initial velocity conditions.

velocity v = rate of disappearance of a substrate, or rate of formation of a

product (more precise)
Why initial velocity?
(1)
(2)
(3)
(4)

The possibility of back-reaction

Denaturation of the enzyme
Product inhibition
Decrease of substrate concentration below the saturation level (can be
avoided with substrate concentration > 4 KM)

The assay should always be tested to show that the velocity v is proportional to the
amount of enzyme added.

[Example] For the determination of the enzymatic activity of alcohol oxidase (AO)
from yeast, the following coupled reactions are utilized:
Methanol O 2

AO formaldehyde H 2 O 2

H 2 O 2 DH 2 HP 2H 2 O D

where DH2 is a dye precursor, D is the dye, and HP is the enzyme horseradish
peroxidase. The dye is monitored by means of a spectrophotometer at a wavelength
of 405 nm. The standard curve of the absorbance versus concentration of H2O2 was
found to be:
A405 0.0377C H 2 O 2 0.0123 (where C H 2 O 2 in mol/L)

A sample of clarified yeast cell lysate assayed for AO activity by this method gave a
rate of change of A405 of 0.082/min after a dilution of 1:40,000. Calculate the AO
concentration in units per mL, where 1 unit (U) is equal to the amount of enzyme
producing 1 mol of H2O2 per minute under the conditions of the assay.
Solution:
A405 0.0377C H 2 O 2 0.0123 or A405 0.0377C H 2O 2

AO activity in the assay

C H 2 O 2
t

A405 / 0.0377
0.082 min -1
mol
U

2.175
2.175
L
t
L - min
L
0.0377
mol

AO activity in the clarified cell lysate = 40000 2.175 U/L = 87 U/mL

#
[Example] To measure the amount of glucoamylase in a crude enzyme preparation, 1
mL of the crude enzyme preparation containing 8 mg protein is added to 9 mL of a
4.44% starch solution. One unit of activity of glucoamylase is defined as the amount
of enzyme which produced a mol of glucose per min in a 4% starch solution at pH
4.5 and 60C. Initial rate experiments show that the reaction produces 0.6 mol of
glucose/mL-min. What is the specific activity of the crude enzyme preparation?
Solution:
Specific activity

(0.6 units/mL) (10 mL)

0.75 units/mg protein
8 mg protein

MICHAELIS-MENTEN KINETICS
k1

E+S

k-1

ES

k2

E+P

Assumptions for the Michaelis-Menten kinetics:

(1) The ES complex is established rather rapidly.
(2) The rate of the reverse reaction of the second step is negligible.
It often holds only when product accumulation is negligible at the
beginning of the reaction.
Approaches used in developing the rate expression: (1) rapid-equilibrium
approach, and (2) quasi-steady-state approach.

A. Rapid-Equilibrium Approach
[E] and [ES] are in equilibrium (it implies that k2 << k1).
[E] [S] k 1

K m (the Michaelis-Menten constant)

[ES]
k1
v

k [ E ][S]
d [ P]
d [S]

k 2 [ ES] 2
dt
dt
Km

[E] and [ES] are difficult to measure.

[E]t = initial amount of enzyme [E] [ES]

[ES]

[E]t
K
1 m
[S]

v k 2 [ ES]

Km
K

[ES] [ES] [ES] 1 m

[S]
[S]

k 2 [ E ]t k 2 [ E] t [S] Vmax [S]

Km
K m [S] K m [S]
1
[S]

where Vmax = maximum reaction velocity = k2[E]t

B. Quasi-Steady-State Approach (Briggs-Haldane Analysis)

E+S

k1
k-1

k2

ES

E+P

[E] and [ES] remain constant.

In the equilibrium approach,
v1 = v1
In the steady-state approach,
v1 = v1 + v2

d [ ES]
k1 [ E ][S] ( k 1 k 2 )[ ES] 0
dt

k k2

[E] t [ E ] [ ES] [ ES] 1

1
k1 [S]

v k 2 [ ES]

[E]

k 1 k 2
[ ES]
k1 [S]

[ES]

[E] t
k 1 k 2
1
k1 [S]

k 2 [ E]t [S]
V [S]
max
k 1 k 2
[S] K m [S]
k1

where Vmax = k2[E]t and K m

k 1 k 2
k1

1
(Note: K m k in the equilibrium approach.)
1

* The steady-state assumption is satisfactory as long as [S]/[E]t > 103.

DETERMINATION OF Km AND Vmax

E+S

k1
k-1

ES

k2

E+P

Vmax [S]
K m [S]

v: initial rate (to avoid product inhibition effect)

v

d [S] d [ P]

dt
dt

* Note: keep [S] < 0.05[S]0 when determining the reaction rate.

A. Lineweaver-Burk Plot:

K
1
1
1
m

v Vmax [S] Vmax

x-axis intercept:
0

Km 1
1

Vmax [S] Vmax

B. Hanes-Woolf Plot
K
[S]
1

[S] m
v
Vmax
Vmax

1
1

[S]
Km

C. Eadie-Hofstee Plot
vKm + v[S] = Vmax[S]
v[S] = Vmax[S] vKm
or v Vmax K m

v
[S]

 Comments on the methods used to determine Km and Vmax:

[S]: controlled by the investigator, quite precisely
v: subject to more or less experimental error
Various linear transformations do not provide equally accurate estimates
of the parameters.
(1) Plot

1
1
v.s.
v
[S]

(Lineweaver-Burk plot)

* Put considerable emphasis on rate values obtained at low [S].

Small v Large

1
v

Important in determining the position of the fitted line.

* Larger errors are expected for smaller vs.
* Least reliable, in general.
(2) Plot

[S]
v

v.s. [S] (Hanes-Woolf plot)

* Give less variation in accuracy over the practical range of [S].

* Put considerable emphasis on small v.
(3) Plot v v.s.

v
[S]

* Place equal emphasis on all points (v appears on both sides).

* Show some degree of inevitable correction.
* Best linear transformation when the error of v is large.

[Example] The following data has been obtained for two different initial enzyme
concentrations for an enzyme-catalyzed reaction.
v at [E]t = 0.015 g/L
(g/L-min)
1.14
0.87
0.70
0.59
0.50
0.44
0.39
0.35

[S]
(g/L)
20.0
10.0
6.7
5.0
4.0
3.3
2.9
2.5

v at [E]t = 0.00875 g/L

(g/L-min)
0.67
0.51
0.41
0.34
0.29

Find Km and k2.

Solution:
K
[S]
1

[S] m
v
Vmax
Vmax

(Hanes-Woolf plot)

From the plot of [E]t = 0.015 g/L,

Vmax = k2[E]t = 1/0.6 = 1.67 g/L-min; Km = 9.2 g/L.
k2

Vmax
1.67
g

111
[ E ] t 0.015
g enzyme - min

From the plot of [E]t = 0.00875 g/L,

Vmax = k2[E]t = 1.0 g/L-min; Km = 10 g/L.
k2

Vmax
1.0
g

114
[ E ]t 0.00875
g enzyme - min

INTEGRATED MICHAELIS-MENTEN EQUATIONBATCH

KINETICS
v
t

[ S]

[ s ]0

Vmax dt

d [S] Vmax [S]

dt
K m [S]

K m [S]
[S]
d [S] Vmax t K m ln 0 ([S]0 [S])
[S]
[S]

K m [S]0
[S]0 [S]
ln
Vmax
t
[S]
t
1

[S]

Plot t ln [S]0 versus

[S]

1 [S]0 [S]
t

0
max
or t ln [S] K K
m
m

[S]0 [S]
.
t

Slope = 1/Km; intercept = Vmax/Km.

Note: (1) Reaction may be product inhibition. (2) Reaction conditions may change.
(pH change) (3) Reaction may approach an equilibrium position. (S P)
Michaelis-Menten assumptions and reaction scheme are invalid.
[Example] An enzyme reaction follows the Michaelis-Menten kinetic, with Km =
0.03 mol/L and Vmax = 13 mol L1 min1. An incoming substrate has a concentration
of 10 mol/L and a flow rate of 10 L/h. (a) What should be the size of a steady-state
continuous stirred-tank reactor for a 95% conversion? Assume that the enzyme
concentration in the reactor is constant. (b) What should be the size of the reactor if
you employ a plug-flow reactor?
Solution:
(a) Continuous stirred-tank reactor
Material balance on the substrate:
{Rate of input} {Rate of output} = {rate of disappearance}
F [S]0 F [S]

Vmax [S]
Vreactor
K m [S]

[S]0 = 10 mol/L; [S] = 0.5 mol/L (for a 95% conversion)

13 0.5
mol
mol 60 min
Vreactor


0.03 0.5
h
h
min

10 (10 0.5)

Vreactor = 0.13 L

(b) Plug-flow reactor

d [S] Vmax [S]
v

dt
K m [S]

max

[ S]

dt

13 0.03 ln

[ s ]0

K m [S]
[S]
d [S] Vmax K m ln 0 ([S]0 [S])
[S]
[S]

10
(10 0.5)
0.5

= 0.74 min

L
h

Vreactor F 10 0.74 min

0.12 L
h

60 min

ENZYME INHIBITION
Enzyme inhibitions may be irreversible or reversible.
* Irreversible inhibitors (such as heavy metals) form a stable complex with
enzyme and reduce enzyme activity.
May be reversed only by using chelating agents (such as EDTA and citrate).
* Reversible enzyme inhibitions: competitive, noncompetitive, and uncompetitive.

Competitive Inhibition
k1

E+S

ES

k-1

k2

E+P

+
I
KI
EI
Km

k 1 [E] [S]
[E] [I]

; KI
k1
[ES]
[EI]

K
[E] [S]m [ ES]

[EI]

K
K [I]

[E] t [E] [ES] [EI] [ES] m 1 m

[S] K I
[S]

d [ P]
k 2 [ ES]
dt

or

[I]
K [I]
[ E] m
[ ES]
KI
[S] K I

[ES]

K m

[E] t [ S ]
[I ]
[S]
1
K I

k 2 [ E]t [S]
Vmax [S]

[I]
[I]
[S] K m 1
[S] where Vmax = k2[E]t
K m 1
KI
KI

* Vmax is unaffected by competitive

inhibitors, but the apparent Km is
increased.
* A higher level of [S] is required to attain
a given reaction rate.
* The degree of inhibition is reduced as
substrate concentration increases.

Clinical Application of Differential Enzyme Inhibition. Human blood serum

contains a class of enzymes known as acid phosphatases, which hydrolyze
biological phosphate esters under slightly acidic conditions (pH 5.0):

Acid phosphatases are produced by erythrocytes, the liver, kidney, spleen, and
prostate gland (). From the clinical standpoint, the enzyme from the
prostate gland is most important because an increased concentration in the blood is
frequently an indication of cancer of the prostate gland. The phosphatase from the
prostate gland is strongly inhibited by the tartrate ion, while acid phosphatases
from other tissues are not. How can this information be used to develop a specific
procedure for measuring the activity of the acid phosphatase of the prostate gland
in human blood serum?
Answer: Measure the total acid phosphatase activity in the presence and absence of
tartrate ion.

Treatment of Methanol Poisoning. Methanol (wood alcohol), a commercial

solvent once used as an automotive antifreeze, is very toxic and may cause death if
a person swallows as little as 30 mL. The extreme toxicity is due not to the
methanol itself but to its metabolic product, formaldehyde. Methanol is rapidly
oxidized to formaldehyde by the action of the liver enzyme alcohol dehydrogenase:

Part of the medical treatment for methanol poisoning is to give the patient ethanol
(ethyl alcohol), either by mouth or intravenously (), in amounts that would
cause intoxication in a normal individual. Explain why this treatment is effective.
Answer: Ethanol competes with methanol for the active site of alcohol
dehydrogenase.

Noncompetitive Inhibition
Km

E+S

k2

ES

I
KI

E+P

KI
Km

EI + S

Km

EIS

k 1 [E] [S] [EI] [S]

k1
[ES]
[ESI]

KI

[E] [I] [ES] [I]

[EI]
[ESI]

K
[E] [S]m [ ES] ;

[EI]

K [I]
[I]
[I]
[ E] m
[ ES] ; [ESI]
[ ES]
KI
[S] K I
KI

Km
K [I] [I]

1 m

[S] K I K I
[S]

[E] t [E] [ES] [EI] [ESI] [ES]

or [ES]

[E]t [ S ]

[I]
[I]
[S] 1

K m 1
KI
K I

d [ P]
v
k 2 [ ES]
dt

Vmax
[S]
[I]

1
K I

k 2 [ E]t [S]

K m [S]

[I]
[I]
[S] 1

K m 1
KI
KI

Vmax , app [S]

K m [S]

Vmax

where Vmax , app

[I]

K I

* The apparent Vmax is decreased by

noncompetitive inhibitors; Km is
unaffected.
* The degree of inhibition is not a function
of the substrate concentration.

Uncompetitive Inhibition
Km

E+S

k2

ES

E+P

+
I
KI
ESI

Km

[E]

K
[I]

[E]t [E] [ES] [ESI] [ES] m 1

K I
[S]

k 1 [E] [S]
[ES] [I]

; KI
k1
[ES]
[ESI]
Km
[I]
[ ES] and [ESI]
[ ES]
[S]
KI

or [ES]

k 2 [ E]t
k 2 [ E]t [S]
d [ P]
k 2 [ ES]

dt
Km

[I]
[ I]

K m [S] 1

K I
K I
[S]

Vmax

where Vmax , app

[I]

K I

[E]t
Km
[I]

1
K I
[S]

Vmax
[S]

[ I]
1

Vmax , app [S]

K
I

Km
[S] K m , app [S]
[I]

1
K I
Km

and K m , app

[I]

K I

* An uncompetitive inhibitor decreases Vmax and

Km to the same extent.

* Inhibition is increased as substrate

concentration increases.
[S]

 Determination of the type of inhibition:

K

m
Lineweaver-Burk equation: v V [S] V
max
max

Plot

1
1
versus
.
v
[S]

y-axis intercept

1
Vmax

; x-axis intercept

1
Km

Vmax is unaffected but Km is increased.

Competitive inhibition.

Km is unaffected but Vmax is decreased.

Noncompetitive inhibition.

Both Vmax and Km are varied.

Uncompetitive inhibition.

VI

Kelompok Bioproses C;
1. Khairull Fahmi
2. Mhd.Reza Rahmadhan
3. Ariel Seumupa
4. Devi Sahara
5. Afdal Supi
6. M. Zulfadli
7. Christina Eka
8. Florentina Pandiangan

[Example] The hydrolysis of urea by urease is an only partially understood reaction

and shows inhibition. Data for the hydrolysis of the reaction are given as follows.
[S] = 0.2 M
1/v
[I]
0.22
0
0.33
0.0012
0.51
0.0027
0.76
0.0044
0.88
0.0061
1.10
0.0080
1.15
0.0093

[S] = 0.02 M
1/v
[I]
0.68
0
1.02
0.0012
1.50
0.0022
1.83
0.0032
2.04
0.0037
2.72
0.0044
3.46
0.0059

where v = mol/L-min and [I] is inhibitor molar concentration. Determine the kinetic
parameters (Km and KI).
Solution:

K
1
1
1
m

(Lineweaver-Burk plot)
v Vmax [S] Vmax

Using the data of [I] = 0, 0.0012, 0.0044, and 0.006 M,

1/Km = 13, or Km = 7.77 102 M; 1/Vmax = 0.2, or Vmax = 5.0 mol/L-min.

The reaction belongs to a noncompetitive inhibition.
For [I] = 0.0012 M and [S] = 0.2 M, v = 3 mol/L-min.

Remarks:

Vmax
5 0 .2
[S]
[I]

0.0012

1
1

KI = 6.0 103 M.
KI
K
I

3
2
K m [S]
7.77 10 0.2

Vmax , app

Vmax

[I]

max
1
or V
KI
max , app

[I]
1

K I

max
versus [I]; slope
.
Plot V
KI
max , app
[I]
0
0.0012
0.0044
0.0060
Vmax,app
5
4
2
1.33
Vmax
1
1.25
2.5
3.76

Vmax,app

4.0
3.5
3.0
2.5

Vmax
Vmax,app 2.0
1.5
1.0
0.5
0

[I] 103, M

Slope

1
0.45
KI

or KI = 2.2 103 M
#

[Problem] The steady-state kinetics of an enzyme is studied in the absence and

presence of an inhibitor (inhibitor A). The initial rate is given as a function of
substrate concentration in the table below.
v (mmol L1 min1)
No inhibitor
Inhibitor A
1.72
0.98
2.04
1.17
2.63
1.47
3.33
1.96
4.17
2.38

[S] (mmol L )
1.25
1.67
2.50
5.00
10.0

(a) What kind of inhibition is involved? (b) Determine Vmax and Km in the absence
and presence of inhibitor.
Solution:
Eadie-Hofstee plot:

v Vmax K m

Plot v versus

v
[S ]

v
[S]

, slope = Km, intercept = Vmax.

4.5
4.0

v, mmol/L-min

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

0.2

0.4

0.6

0.8

v/[S], min

1.0

1.2

1.4

1.6

() In the absence of inhibitor, Km = 2.5 mmol L1, Vmax = 5.0 mmol L1 min1.
() With inhibitor, Km, app = 2.5 mmol L1, Vmax, app = 3.0 mmol L1 min1.
It is of noncompetitive inhibition.
#

KINETICS FOR REVERSIBLE REACTIONS

Glucose
E+S

glucose isomerase

k1
k-1

ES

fructose

k2

E+P

k-2

By steady-state approximation,
d [ ES]
k1 [S][ E ] ( k 1 k 2 )[ ES] k 2 [ E ][ P] 0
dt

1
2
or [ E] k [S] k [ P] [ ES]
1
2

k 1 k 2

[ E ]t [ ES] [ E ] [ ES] 1
k1[S] k 2 [ P]

k [ P] ( k 1 k 2 )
d [S] d [ P]

k 2 [ ES] k 2 [ E][ P] k 2 2
[ ES]
dt
dt
k1 [S] k 2 [ P]

k 2 [ P] ( k 1 k 2 )
[ E]t
k1 [S] k 2 [ P]
k k [S] k 2 k 2 [ P] k 2 [ P] (k 1 k 2 )[ E]t
1 2
k 1 k 2
k1 [S] k 2 [ P] k 1 k 2
1
k1 [S] k 2 [ P]

k2

k1k 2 [S] k 1k 2 [ P]

[ E]t
k 1 k 2
k 1 k 2

k1 [S]
k [ P]
2
1
k 1 k 2 k 1 k 2
Set

k 1 k 2
k k2
K S ; 1
K P ; k 2 [ E ]t VS ; k 1 [ E] t VP
k1
k 2

VS
V
[S] P [ P]
K
K
v S [S] [PP]
1

KS KP

ALLOSTERIC ENZYME KINETICS

Some enzymes have more than one substrate binding site. The binding of one
substrate to the enzyme facilitates binding of other substrate molecules.
k1

E + nS
[E] [S]n
Km
[ES n ]

[E]

k-1

ESn

Vmax [S]n
v
vK m v[S]n Vmax [S]n
K m [S]n
vK m (Vmax v )[S]n

ln K m ln

ln

(Vmax v ) n
[S]
v
(Vmax v )
n ln[S]
v

v
(Vmax v )

Km

1 [ES n ]

n
[S]

k 2 [ E]t k 2 [ E]t [S]n Vmax [S]n

n
Km
K

[S]
K m [S]n
m

1
[S]n

* Determination of kinetic parameters:

Km

E + nP

Km
[ES n ] [ E]t [E] [ES n ]
n
[S]

v k 2 [ ES n ]

k2

n ln[S] ln K m

SUBSTRATE INHIBITION
The enzyme, which is probably oligomeric, has multiple binding sites for substrate.
Km

E+S

k2

ES
+
S

E+P

KSI
ES2

ES2 does not react further.

Km

[E] [S]
[ES] [S]
; K SI
[ES]
[ES 2 ]

or [ES]

K
[S]

[E]t [E] [ES] [ES 2 ] [ES] m 1

K SI
[S]

[E]t
Km
[S]

1
K SI
[S]

k 2 [ E]t
Vmax [S]
d [ P]
k 2 [ ES]

dt
[S]2
Km
[S]
K m [S]

1
K SI
K SI
[S]

Remarks:
(1) At low [S], the term involving [S]2 can be neglected, and inhibition effect is not
observed.
v

Vmax [S]
[S]2
K m [S]
K SI

[S]

max
v K [S]
m

(2) At high [S], inhibition is dominant.

v

Vmax [S]
V
max
2
[S]
[S]
1
[S]
K SI
K SI

(3) The substrate concentration resulting in the maximum reaction rate, [S]max, can be
determined by setting dv/d[S] = 0.

dv
d

d [S] d [S]

Km

Vmax [S]
[S]2
[S]
K SI

[S]max

K m K SI

BISUBSTRATE REACTIONS: SINGLE-DISPLACEMENT

REACTIONS
Direct transfer of an atom or a functional group from one of the substrate to the
other.

KA

[EB]

[E] [A] [EB] [A]

[E] [B] [EA] [B]

; KB

[EA]
[EAB]
[EB]
[EAB]

KA
K
K
K K
[EAB] ; [EA] B [EAB] ; [E] A [EA] A B [EAB]
[A]
[B]
[A]
[A] [B]

K AK B K B K A

1 [EAB]
[A] [B] [B] [A]

[E]t [ E] [EA] [EB] [EAB]

k 5 [E]t
Vmax [A] [B]
d [ P]
k5 [ EAB]

K AKB KB K A
dt

1 K AK B K B [ A ] K A [ B] [ A ][ B]
[A] [B] [B] [A]

where Vmax = k5[E]t

BISUBSTRATE REACTIONS: DOUBLE-DISPLACEMENT (OR

PING-PONG) REACTIONS

AX donates X to the enzyme.

A must then leave before B,
the acceptor of X, is bound.
Occurs when coenzymes are
involved.

E + AX

EX + B

k1

k2

EAX

k-1
k3

k4

EBX

k-3

d [ EBX ]
k 3 [EX] [B] ( k 3 k 4 ) [EBX] 0
dt

d [ EAX ]
k1 [E] [AX] ( k 1 k 2 ) [EAX] 0
dt

[E]

E + BX

[EX]

[EAX]

k 2 [EAX] k 4 [EBX]

EX + A

k 3 k 4
[ EBX ]
k 3 [B]

k4
[EBX]
k2

( k 1 k 2 )
( k k 2 )k 4
[ EAX ] 1
[ EBX ]
k1 [AX]
k1k 2 [AX]

( k 1 k2 )k4 k4 k 3 k4

1 [EBX]
k2
k3[B]
k1k2 [AX]

[E]t [ E] [EAX] [EX] [EBX]

v k 4 [EBX]

k 4 [E]t
Vmax

( k 1 k 2 )k 4 k 4 k 3 k 4
K
K

1 Km A B
[ AX ] [ B]
k1k 2 [AX] k 2
k 3 [B]

where Vmax k 4 [ E ]t ; K m 1

k4
( k k 2 )k 4
k k4
; K A 1
; K B 3
k2
k1k 2
k3

EFFECT OF TEMPERATURE ON ENZYME ACTIVITY

Enzymes have an optimal temperature for their activities.

* For many proteins, denaturation begins to occur at 45 to 50C and is severe at

55C.
* In nearly all cases, inactivation becomes virtually instantaneous at
temperature well below 100C.
* Thermophilic bacteria contain heat-stable enzymes.
A crystalline -amylase from Bacillus stearothermophilus can retain
90% of activity after 1 h at 90C.
The effect of temperature on enzyme activity may be due to several different
causes:
(1) The actual velocity of breakdown of the enzyme-substrate complex
(2) The stability of enzyme
(3) Change of the affinity of the enzyme for substrate (Km), activators, or
inhibitors
(4) Change of the rate-limiting step
(5) Change of the oxygen solubility
(6) Change in the pH of the buffer used
Both catalytic and denaturation reactions can be represented by the Arrhenius
equation:
k Ae E / RT

where k = reaction rate constant, A = Arrhenius constant, E = activation energy, R

= gas-law constant, and T = absolute temperature.

ln k ln A

E
RT

Plot ln v versus

1
E
, slope
T
R

For enzyme-catalyzed reactions, Ea = 420 kcal/mol (with most values about 11

kcal/mol); for enzyme deactivation, Ed = 40130 kcal/mol (with most values about
70 kcal/mol).
ln

kT 2
E 1
1


kT 1
R T2 T1

* A rise in temperature from 3040C,

1.8-fold increase in the rate of catalysis (for Ea = 11 kcal/mol), while 41fold increase in the rate of denaturation (for Ed = 70 kcal/mol).
The temperature range of Arrhenius relationship is quite limited.

EFFECT OF pH ON ENZYME ACTIVITY

Proteins possess many ionizable groups.
pH change will affect the catalytic site and conformation of the enzyme.
In general, enzymes are active only over a limited pH range, and in most cases a
definite optimal pH is observed.

The optimal pH may be due to:

(1) A true reversible effect of pH on Vmax
(2) A change in the enzyme/substrate affinity
(3) A change in the stability of the enzyme
Enzymes may become irreversibly denatured at the extremes of pH.
The optimal pH is a function of other parameters such as temperature, substrate
concentration, and buffer solution.
pH dependence of the reaction rate for ionizing enzymes:
E- + H+

Km

K2
EH + S
+
H+

Km

EHS

k2

EH + P

[EH] [S]
[EH] [H ]
[E - ] [H ]
; K1
;
K

2
[EHS]
[EH 2 ]
[EH]

[E]t = [E] + [EH] + [EH2+] + [EHS]

v k 2 [EHS]

K1
EH2+

Vmax [S]

K
[H ]
[S]
K m 1 2
[H ] K1

or v

Vmax [S]
K
[H ]

where K m ,app K m 1 2
K m , app [S]
K 1
[H ]

 Rate expression for the case of ionizing substrate:

+

SH + E

Km

ESH

k2

K1

E + HP

Km

[SH ] [E]
[S] [H ]
;
K

1
[ESH ]
[SH ]

[E]t = [E] + [ESH+]

S + H+

v k 2 [ESH ]

Vmax [S]

K
K m 1 1 [S]
[H ]

Theoretical prediction of the pH optimum of enzymes requires knowledge of the

active site characteristics.
This is very difficult to obtain.
The pH optimum is usually determined experimentally.

VII
Kelompok bioprocess kelas C :
1. Gewa Handika (13-082)
2. Sy. Nadiratuzzahrah (13-083)
3. Yayang Afandy (13-084)
4. Muhammad Ridha (13-085)
5. Finnya Angkie Winoto (13-087)
6. Irsa Septiawan (13-100)
7. M. Faizul Hisham (13-103)
8. Salwa Jody Gustia (13-104)

EFFECT OF BUFFER ON ENZYME REACTION

A buffer system consists of a weak acid (the proton donor) and its conjugate base
(the proton acceptor).
* Buffering action:
H+ + A-

HA

Ka

[H ][A - ]
[HA]

or

[H ] K a

[HA]
[A - ]

where Ka = acids dissociation constant

If the concentrations of the buffer components (i.e., [HA] and [A]) are
made equal, the solution will be buffered at a pH corresponding to logKa.
* Titration curves of weak acids:

 If the concentrations of the buffer components are higher than added acid
(or base), the buffer ratio (and so the pH) will change little.

 Two examples of preparation of buffers:

 The choice of buffer system is a very important parameter in enzyme reactions.

* The nature of the buffer system may influence the enzyme reaction.

* The nature of the buffer may play an important role in enzyme stabilization.
It is commonly observed that carboxylic and sulfonic acid buffers stabilize
enzymes.
Tris buffer has been known to cause a significant number of problems.
* Ionic strength may have a strong effect on enzyme stability and activity.

* Buffers may influence the thermodynamics of the catalyzed reaction.

O
H2N C NH2 + H2O + H+

urease

HCO3- + 2NH4+

The true product of the urease reaction is ammonium carbamate, which is

decomposed by a subsequent buffer-mediated proton transfer.

EFFECT OF ENZYME CONCENTRATION ON REACTION RATE

In most cases, any two molecules of enzyme function independently, i.e., v [E]t.
Other proportionalities are due to:
(1) Inhibitors are present in the assay solution.
(2) Insufficient amount of coenzyme added.
(3) The enzyme reaction is not the rate-limiting step.
Instead, it is the diffusion of macromolecular substrate.
* Examples:
Lipases act on triglycerides (macromolecular substrate)
v [E]t1/2
Lipases act on benzyl ethyl ester (small substrate)
v [E]t
Deviations from linearity with enzyme concentration:
A: normal cases.
B: insufficient substrate to saturate the
enzyme at high [E]t.
C: an inhibitor for which the enzyme has
high affinity is present in the substrate
solution.
D: an inhibitory substrate is present in the
enzyme solution.

ENZYME DEACTIVATION
Protein structure is stabilized by weak forces.
Forces that maintaining protein structure: hydrogen bonding, ionic attractions,
hydrophobic interactions, and S-S linkages.
Native protein structure is only marginally stable.
Stages of protein unfolding:

N = native conformation
A* = critically activated state
ID = incompletely disordered conformation
RC = random coil (fully denatured state)

There are many factors that can cause reduction in enzyme activity.
It is not the individual factors but their combinations that determine rates of
enzyme deactivation.
The protein denaturants can be classified into physical, chemical, and
biological denaturants.
(1) Physical denaturant
Heat
Target: hydrogen bonds
Driving force: increase of denatured conformations due to increased
thermal movement and decreased solvent structure; irreversible
covalent modification (e.g., disulfide interchange)
End product: highly disordered conformation; aggregates
Cold
Target: hydrophobic bonds; solvated groups
Driving force: altered solvent structure; dehydration
End product: aggregates; inactive monomers

 Mechanical forces
Target: solvated groups; void volume
Driving force: changes in solvation and void volume; shearing
End product: highly disordered conformation; inactive monomers
Radiation
Target: functional groups (e.g., cySH, peptide bonds)
Driving force: decrease of structure-forming interactions after photooxidation or attack by radicals
End product: highly disordered conformation; aggregates
(2) Chemical denaturant
Acids
Target: buried uncharged groups (e.g., his, peptide bonds)
Driving force: decrease of structure-forming ionic interactions
End product: random coil
Alkali
Target: buried uncharged groups [e.g., tyr, cySH, (cyS)2]
Driving force: decrease of structure-forming ionic interactions
End product: random coil
Organic H-bond-formers
Target: hydrogen bonds
Driving force: decrease of structure-forming H bonds between water
and native conformation
End product: random coil
Salts
Target: polar and nonpolar groups
Driving force: salting in/salting out bias of polar and nonpolar groups
in solvent
End product: highly disordered conformation
Solvents
Target: nonpolar groups
Driving force: solvation of nonpolar groups
End product: highly disordered conformation; peptide-chains with
large helical regions

 Surfactants
Target: hydrophobic domains (all surfactants) and charged groups
(ionic surfactants only)
Driving force: formation of partially unfolded structures including
micelle-like regions
End product: incompletely disordered conformation; large helical
regions
Oxidants
Target: functional groups (e.g., cySH, met, try, and others)
Driving force: decrease of structure-forming and/or functional
interactions
End product: inactivated enzyme; sometimes disordered structure
Heavy metals
Target: functional groups (e.g., cySH, his, and others)
Driving force: masking of groups pertinent to structure or function
End product: inactivated enzyme
Chelating agents
Target: cations important for structure or function
Driving force: ligand substitution or cation removal
End product: inactivated enzyme
(3) Biological denaturant
Proteases
Target: peptide bonds
Driving force: hydrolysis of peptide bonds
End product: oligopeptides, amino acids

 The simplest model of enzyme deactivation is irreversible first order kinetics.

EN

kd

ED

EN = native enzyme; ED = denatured enzyme.

[E ]
d [E N ]
k d [E N ] ; I.C. : [E N ] [E]0 at t 0 ln N k d t
[E]0
dt

* Obtaining the enzyme deactivation data:

Expose the enzyme to denaturing conditions for some time interval in
the absence of substrate, and then making an initial rate activity assay
after returning the enzyme solution to the standard conditions.
* Data for the deactivation of ATPase under several different conditions are
consistent with ln([EN]/[E]0) = kdt.

Decay of enzyme activity with time does not always follow the first-order model.
* The profile of luciferase deactivation contains two different linear regions.
A model that fits the data of
luciferase deactivation is:
ED1

kd1
kr
EN
kd2

ED2

d [E N ]
(k d 1 k d 2 )[E N ] k r [E D1 ]
dt
d [E D1 ]
k d 1 [E N ] k r [E D1 ]
dt

I.C.: t = 0, [EN] = [E]0 and [ED1] = 0

* Loss of activity in protease solution includes deactivation and autodigestion.

EN

EN + ED1

ED1
Km

kd1

ED2

ENED1

kd2

EN + hydrolysis products

* Irreversible enzyme deactivation by a poison:

EN + poison ED
rd

d [E N ]
k d [ E N ][ poison]
dt

Combination of deactivation model with catalytic reaction sequence:

EN + S

k1
k-1

ENS

k2

EN + P

kd
ED

* ENS is not subjected to deactivation.

[ E N ][S] k 1
[S]
[E N ]

K m [ E N S]
Km
[ E N S]
k1

[S]
[S]

[ E N ]t [ E N ] [ E N S] [ E N ]
[ E N ] [ E N ] 1
Km
K m

[E N ]

[ E N ]t
[S]
1
Km

d [ E N ]t
k [E ]
d ln[E N ]t
kd
k K
k d [ E N ] d N t

d m
[S]
[S] or
dt
dt
K m [S]
1
1
Km
Km

STRATEGIES FOR ENZYME STABILIZATION

Native enzymes are subject to inactivation by chemical, physical, and biological
factors, and the inactivation can occur either in storage or during use.
Physical factors that inactivate enzymes: shear force, surface tension
* Shear denaturation could be correlated as a function combining shear rate
and exposure time.

* Data of surface tension: 80 dyne/cm for air-water interface; 1 dyne/cm for

plasma membrane of the cell.
Need surfactants to lower the air-liquid surface tension in the
operation of foam fractionation.
Methods for enzyme stabilization:
(1) Addition of stabilizing compounds
Substrate
The active site of an enzyme may be its least stable region.
* -Amylase is stabilized by addition of starch.

* Glucose isomerase is stabilized against heat damage by addition of

glucose.
Organic solvents
There are many cases that addition of organic solvents cause
denaturation at high concentrations but afford considerable
stabilization at low concentrations.
A possible rationale: prevention of hydrogen bond rupture.
Salts
Cations such as Ca, Cu, Fe, Mn, Mo, and Zn have an effect on the
stability and activity of metalloenzymes.
* Calcium helps to stabilize the tertiary structure of -amylase of
Bacillus caldolyticus.
Ca2+ can serve as a stabilizing bridge analogous to a disulfide
bond.
Polymer additives
Natural or synthetic polymers such as gelatin, albumin, or
polyethylene glycols can increase the heat stability of enzymes.
(2) Chemical modification of enzymes
Crosslinking with bifunctional reagents such as glutaraldehyde
To limit access by proteases and may also serve to lock the protein
into an active configuration.
Adding polyamino acid side chains to the R-groups of certain amino acid
residues
Use acylation or alkylation to introduce other substituents.
(3) Immobilization of enzymes
Immobilized enzymes are often more stable than enzymes in solution
Examples of enzyme stabilization:
* Glucoamylase
Methods: addition of substrate analogs, glucose, gluconolactone
Effect: enhanced thermal stability
* Lactate dehydrogenase
Methods: addition of substrate lactate or effector fructose-diphosphate
Effect: greater thermal stability
Note: the enzyme is destabilized by addition of pyruvate substrate.

* -Amylase
Method: addition of 5070% sorbitol
Effect: better storage and thermal stability
* -Amylase (Bacillus caldolyticus)
Method: addition of Ca2+
Effect: greatly enhanced thermal stability
* Chymotrypsin
Method: addition of 5090% glycerol
Effect: improved resistance to proteolysis
* -Galactosidase
Methods: addition of 510% ethanol or 2-propanol
Effect: increased heat stability
Note: similar levels of methanol, n-propanol destabilize the enzyme.
* Trypsin
Method: addition of polyalanyl (~10 units long) to proteins amino groups
Effect: more proteolysis, heat deactivation resistance
* Asparaginase
Methods: succinyl substituents added using the acid anhydride
Effect: increased protease resistance
* Glycogen phosphorylase
Methods: butyl or propyl substituents added using the aldehyde and NaBH4
Effect: enhanced thermal stability
* Papain
Method: cross-linked using glutaraldehyde
Effect: better thermal stability
Comments on methods for enzyme stabilization:
(1) The influence of a particular chemical or strategy on one enzymes stability
cannot always be extrapolated to other enzymes.
(2) The stabilizing effect depends often on a particular combination of solution
composition and temperature.