Biochemical Engineering

7RCHE31

Dr. Sudhir Ranganath

Contact Hours/Week: 3 (Lecture) Credits: 3.0

CIE Marks: 50
Total Lecture Hours: 39 SEE Marks: 50
UNIT III
ENZYME CATALYZED REACTIONS
Mechanism & Michealis-Menten kinetics model
& estimation of kinetic parameters

Batch & continuous enzyme reactor kinetics

Multi-substrate enzyme reaction kinetics & estimation of

kinetic parameters

Kinetics of enzyme inhibition & estimation of kinetic

parameters

Effects of temperature, pH & shear

Immobilized enzymes
Enzyme Inhibition
Certain molecules inhibit enzyme activity
Structural analogs to substrate: Reversible or irreversible
Inhibitors bind to other portion of the enzyme
Irreversible Inhibitors
Covalent binding of the inhibitor to the active site
Prevents S binding
Destroys part of active site
Reduce effective [E] but not the kinetics w.r.t. [S]
Eg: Aldehydes, Alkenes, Phenyl Sulfonates, etc.
Reversible Inhibitors
Weak non-covalent binding of the inhibitor to active site or elsewhere
Removed using either more S or chelating agents (EDTA, citrate)
Upon removal of the inhibitor, enzyme activity is restored
Eg: Protease inhibitors, Ethanol, etc.
Three classes of reversible enzyme inhibitions
Competitive inhibition
Non-competitive inhibition
Uncompetitive inhibition
Substrate inhibition
Competitive Inhibition
Inhibitor is structurally similar to S & competes with S for active site binding
& prevents S binding
Combines with E to form complex (dead-end complex)
Observed kinetics depend on [I] and [S]
Reaction products can also be competitive inhibitors
Many chemotherapeutic drugs are competitive inhibitors

The competitive inhibition scheme can be described as follows:

k1 k2
E + S ES E+P
+ k-1
I Let us assume rapid equilibrium, which gives:

KI % ['] % [)]
#
!" = [%']
and !)# = [%)]
EI
and, *+ = * + *- + [*.] and / = 01 [*-]
Competitive Inhibition
The following equation for the rate of enzymatic conversion is developed:

#$ [&] 01 [2]
!= [,]
→ / = 3)
) *+
($ +[&] 1,566 +[2]
- ,

; ; 1+ [>]
where, 78,9:: = 78 [I] > 0
(,

The net effect of competitive

[I] = 0
inhibition is an increased value of
;
78,9:: and, therefore, reduced 1/V
reaction rate.
1/Vm
Competitive inhibition can be
overcome by high concentrations
of substrate.
-1/Km -1/Km,app 1/[S]
Noncompetitive Inhibition
Noncompetitive inhibitors are not substrate analogs
These inhibitors bind on sites other than the active sites
They bind either to E or ES and reduce the affinity to the substrate
Heavy metals are examples

The noncompetitive inhibition scheme can be described as follows:

K’m k2
E +S ES E+P
+ + With the definitions of:
I I
# % ['] %) [']
!" = = and
KI KI [%'] [%')]

K’m !) =
% [)]
=
%' [)]
EI + S ESI [%)] [%')]

*+ = * + *- + *. + [*-.] and / = 01 [*-]

Noncompetitive Inhibition
The following equation for the rate of enzymatic conversion is developed:

#$ /0,233 /0,233 [6] /0

!= [(] *+$
→.= 5+0
= 5+0 &[6]
where, /0,233 = [(]
%&* %& [,] 4& [6] %&*
(
(

The net effect of noncompetitive

inhibition is a reduction in 78 . High
substrate concentrations would not [I] > 0
overcome noncompetitive
inhibition.
[I] = 0
Other reagents need to be added to 1/V
block binding of the inhibitor to the
enzyme. 1/Vm,app

In some forms of noncompetitive 1/Vm

: is
inhibition, 78 is reduced and 98
increased. This occurs if the -1/Km,app
-1/Km 1/[S]
complex ESI can form product.
Uncompetitive Inhibition
Uncompetitive inhibitors bind only to ES complex and not to free E
The inhibitors have no affinity to the enzyme itself
HIV drug Nevirapine is an example

The scheme for uncompetitive inhibition is as follows:

K’m k2
E +S ES E+P
+
I
KI
With the definitions of:

ESI # =
!"
% [']
and !) =
%' [)]
[%'] [%')]

*+ = * + *- + [*-.] and / = 01 [*-]

Uncompetitive Inhibition
We can develop the following equation for the rate of reaction:

$% ['] .
-% 34,677 [8]
!= # + [0] → 2=
[+]
)*,
[+]
)*, 9.4,677 *[8]
+ +

The net effect of uncompetitive

inhibition is a reduction in both
:; and <; = values. [I] > 0

Reduction in :; has a more

1/V [I] = 0
pronounced effect than reduction in
<;= , and the net result is a

reduction in reaction rate. 1/Vm,app

Uncompetitive inhibition is
described in the figure.
-1/Km,app -1/Km 1/[S]
Substrate Inhibition
High substrate concentration may cause inhibition in some enzymatic
reactions … called substrate inhibition.
S binds to an alternative site (which is other than the active site)
This results in an unreactive ES
Eg: Substrate inhibition of phosphofructokinase

The reaction scheme for uncompetitive substrate inhibition is as follows:

K’m k2
E +S ES E+P
+
S
!"#

ES2
Substrate Inhibition
Consider the following equations:

" [&"] * = " [&]

!"# = [&"( ]
and !) [&"]

,- ["]
The assumption of equilibrium gives: + = [1](
/
.- 0 " 0
21
#
The double-reciprocal plot describing
substrate inhibition is given below
using the above equation:

["](
At low [S], the value of ≪ 1, and
.1#
hence inhibition effect is not seen and
the resulting equation is same as the
M-M kinetics equation.
Substrate Inhibition
$ ⁄ % ≪ 1, and the inhibition is very high
At high substrate concentrations, "#
(dominant). The rate is given by:

*+ , , [5]
(= or.
3
=* +6
,-
[/] + /2 *+
1/
2

A plot of 1⁄( versus [%] results in a line of slope 1⁄ "52 7# and intercept
1⁄7# .

The substrate concentration resulting in the maximum reaction rate can be

83
determined by setting
8[5]
= 0.

Based on this, the % #:; is given by: < =>? = @$=. @<B
Problem 11
Decarboxylation of Glyoxalate by mitochondria is inhibited by malonate. Using
the following data obtained in batch experiments, determine the following

Glyoxalate Rate of CO2 evolution (mmol/L.h)

(mM) I=0 I = 1.26 mM I = 1.95 mM
0.25 1.02 0.73 0.56
0.33 1.39 0.87 0.75
0.40 1.67 1.09 0.85
0.50 1.89 1.30 1.00
0.60 2.08 1.41 1.28
0.75 2.44 1.82 1.39
1.00 2.50 2.17 1.82

a. The type of inhibition.

b. Constants Vm, Km’ and KI.
Solution to Problem 11a
Plot 1/S vs 1/v
Notice that Vm is the same for all three cases. However, Km’ increases as I
increases to Km,’app resulting in a reduction in reaction rate. This is
characteristic of competitive inhibition.
Solution to Problem 11b
Determination of K’m, Vm and KI

First, find out K’m, Vm

! !
For I = 0, from the graph we have
"
= 0.194 )
+ 0.1578, so

1/Vm = intercept = 0.1578 and Vm = 6.34 mM/h

34 5
./012 = 65
= 0.194, so, K’m = 1.23 mM

To find KI, we need to find K’m,app at both Inhibitor concentrations

385,9::
At I = 1.26 mM, ./012 = , K’m,app = 1.96 mM
65

Similarly, at I = 1.95 mM, K’m,app = 2.63 mM

Solution to Problem 11b
*
For competitive inhibition, we have !′#,%&& = !′# ( + !
*

At I = 1.26 mM, we get KI = 2.16 mM

At I = 1.95 mM, we get KI = 1.71 mM

Do you notice any aberration in KI values??

Problem 12
The following data were obtained from enzymatic oxidation of phenol by
phenol oxidase at different phenol concentrations.

S (mg/L) 10 20 30 50 60 80 90 110 130 140 150

v
5 7.5 10 12.5 13.7 15 15 12.5 9.5 7.5 5.7
(mg/L.h)

a. What type of inhibition is this?

b. Determine the constants Vm, Km’ and KSI.
c. Determine the oxidation rate at [S] = 120 mg/L and
Solution to Problem 12a
Plot S vs v
Presence of Smax and the nature of curve suggests that this is substrate
inhibition.
Solution to Problem 12b
To find Vm and K’m plot 1/S vs 1/v
#$
at low [S], inhibition is not in effect, and ! = '($ and
%&
)
% % ,-$ %
*
=# + #$ .
$

Vm = 20.27 mg/L.h

K’m = 31.1 mg/L

y = 1.5355x + 0.0493
KSI ???
Solution to Problem 12b and c
&' "
To find !"# we use $ = + , ---------------- (1)
()' * " *
-+
#

./
To determine !"# , set =0 and solve (1), we get
. "

1 234 = 5′2517

From the plot, 8 9:; = 90 mg/L ….. So, 517 = 260.5 mg/L

(c) Find rate of reaction at S = 120 mg/L

At this S, do we observe substrate inhibition??? Yes

Substituting Vm, K’m and KSI, into (1) we get v = 11.9 mg/L.h
Problem 13
The following table indicates the rates at which a substrate reacts as catalyzed
by an enzyme (1) in the absence of inhibitor; (2) in presence of 10 mM
concentration of the inhibitor. Determine:

[S] Rate of Reaction (MS-1)

(mM) I=0 I = 10 mM
1 1.17 0.77
2 2.1 1.25
5 6.3 2
10 7.6 2.5
20 9 2.86

a. The type of inhibition.

b. Constants Vm, Km’ and KI.
Solution to Problem 13a
Plot 1/S vs 1/v
Notice that Vm is different for I = 0 and I = 10 mM. However, Km is the
same. This is characteristic of non-competitive inhibition.
1.4
I = 0 mM
y = 0.9989x + 0.3001
1.2 R² = 1
I = 10 mM
1
1/v

0.8

0.6

0.4
y = 0.3017x + 0.0987
0.2 R² = 0.99973

0
-0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
-0.2
1/[S]
Solution to Problem 13b
1/Vm = 0.09 for I = 0 and 1/Vm,app = 0.29 for I = 10 mM.

However, -1/Km = -0.3 for both I = 0 and I = 10 mM. This is characteristic of

non-competitive inhibition.

Therefore,
1.4
I = 0 mM
y = 0.9989x + 0.3001
Vm = 11.11 MS-1 1.2
I = 10 mM
R² = 1

1
Vm,app = 3.4482 MS-1 1/v

0.8

Km = 3.33 mM 0.6

Vm,app = Vm (1+[I]/KI) 0.4

1/#$,&'' y = 0.3017x + 0.0987
−1/)$ 0.2 R² = 0.99973
So, KI = 4.5 mM
0 1/#$
-0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
-0.2
1/[S]
Thank you