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2.3 In some enzyme- catalyzed reaction, multiple complexes are involved as follows:
S + E ( ES )1
( ES )1 ( ES )2
( E )2 P + E
REQUIRED: Develop a rate expression using
a. Michaelis Menten approach
b. The Briggs Haldane approach
SOLUTION :
rp=
dcs dcp
=
=ksCesk 4 CpCe
dt
dt
Ces=
Ces=
k 1CsCeo
k 2+k 1 Cs
CeoCs
k2
+Cs
k1
rp=( k 3+k 4 Cp )
CeoCs
k2
+Cs
k1
- k4Ceo
( kk 21 Cs )(k 4 CpCeo)
( ks+ k 4 Cp ) (CeoCs )
rp =
k2
+Cs
k1
k 3CeoCs+k 4 CeoCpCs
rp=
k 2k 4 CpCeo
k 4 CpCesCs
k1
k2
+Cs
k1
k 2 k 4 CpCeo
k1
k2
+Cs
k1
k 3CeoCs
rp=
Ceo k 3 cs
rp=
k 2k 4
Cp
k1
k2
+Cs
k1
Ce=Ceo=Ces
K1Cs ( Ceo-Ces) = k2Ces
K2Ces + k1CsCes = k1CsCeo
k 1 CsCeo
k2
+Cs
k1
CsCeo
k2
+ Cs
k1
ANSWER
k 3 CeoCs rmaxCx
=
k2
Km+Cs
+Cs
k1
Ces=
k 1 CsCeo
k 1 Cs+k 2+k 3
CsCeo
k 3+k 2
+ Cs
k1
dcs dcp
k 3 CsCeo
=
=
dt
dt
k 3+ k 2
+Cs
k1
rp=
rmaxCs
KmCs
Evaluate the Michaelis-Menten kinetic parameters by employing (a) the Langmuir plot, (b)
the Lineweaver-Burk plot, and (c) the Eadie-Hofstee plot.
Given: *refer to table
Required: Michaelis-Menten kinetic parameters
Solution:
a. Langmuir
CS K M 1
=
+
CS
r
r max r max
x, CS
0.0032
0.0049
0.0062
0.0080
0.0095
y, CS/r
0.0032/0.111
0.0049/0.148
0.0062/0.143
0.0080/0.166
0.0095/0.200
b. Lineweaver-Burk
1 1
KM 1
=
+
r r max r max CS
x, 1/CS
1/0.0032
1/0.0049
1/0.0062
1/0.0080
1/0.0095
y, 1/r
1/0.111
1/0.148
1/0.143
1/0.166
1/0.200
y, r
0.111
0.148
0.143
0.166
0.200
c. Eadie-Hofstee
r = r max - K M
x, r/CS
0.111/0.0032
0.148/0.0049
0.143/0.0062
0.166/0.0080
0.200/0.0095
r
CS
@t=5 minutes
Solution:
K M ln
0.01
Cso
+(CsoCs)=r max t
Cs
mol
1
mol
ln
+ (10.9 ) ( 3.4 x 104 )
=r max ( 5 x 60 ) s
L
0.9
L
@t= 15 minutes
K M ln
Cso
+(CsoCs)=r max t
Cs
0.01
Given:
KM = 0.03 mol/L
rmax= 13 mol/ L min
Cso= 10 mol/L
F= 10 L/h
Sol'n:
a)
F 1
rmax Cs
= =
V (CsoCs)(Km +Cs)
Req'd:
a)VCSTR
b) VPFR
V=
F
rmax Cs
(CsoCs)(Km+Cs)
V=
)(
+
)
L
L
L
L
VCSTR=0.1291 L
b)
CsoCs
t
=Km+rmax (
)
Cso
Cs
ln (
)
ln
Cs
Cso
( )
100.5
t
=( 0.03 ) +(13)(
)
10
10
ln
ln
0.5
0.5
( )
( )
t = 0.7377 min
t=V/F
V=Ft
V=(0.7377 min)(1 h/ 60 min)(10 L/h)
VPFR = 0.1229 L
grams
liter
g
Rmax = 7 Lmin
If we operate two 1-L CSTR n series at steady state, wht will be the concentration of substrate
leaving the second reactor? The flow rate is 0.5 L/min. The inlet substrate concentration is 50g/L
and the enzyme concentration in the two reactors is maintained in the sa value all of the time. Is
the two reactor system more efficient than one reactor whose volume is equal to the sum of the
two reactors?
GIVEN:
Cso
50g/L
Km= 10g/L
rmax= 7g/L-min
F= 0.5 L/min
REQUIRED:
a. Cs2
b. Is two reactor more efficient than 1 reactor with volume = 2L
Cs=-km +
rmax Cs
CsoCs
Cs1= (-10g/L) +
50 g
Cs1
L
Cs1= 38.8650g/L
At the second reactor ; cso =38.8650g/L
Cs2= (-10g/L) +
min
38.8650Cs 2
Cs2= 28.50120g/L
Which is more efficient
% conversion =
CsoCs
x 100
Cso
%conversion =
5028.5012
x 100
50
min
50 g
Cs1
L
%conversion=
5029.1517
x 100
50
Rate (mol/L.min)
Presence of Prostigmine
0.059
0.071
0.091
0.111
0.125
0.07
0.06
0.05
Cs/r
0.04
0.03
0.02
0.01
0
0.01
0.01
0.01
0.01
Cs
Linear ()
Linear ()
0.01
0.01
Inhibitor
mol/L
146
146
146
Is this competitive inhibition or noncompetitive inhibition? Justify your answer by showing the
effect of the inhibitor graphically. [Contributed by Professor Gary F. Bennett, The university of
Toledo, Toledo, OH]
Required: MM constants
Without Inhibitor
With inhibitor
a) Langmuir
C s km
1
=
+
(C )
r r max r max s
a) Langmuir
C s km
1
=
+
(C )
r r max r max s
r = 0.9968 mol/L-min
r(max) = 0.4215 mol/L-min
km = 3.6317 mol/L
b) Lineweaver Burk
k
1
1
1
=
+ m ( )
r r max r max C s
r = 0.9916 mol/L-min
r(max) = 0.1567 mol/L-min
km = 2.9807 mol/L
b) Lineweaver Burk
1
1 km 1 1
=
+
( )
r r max r max C s
r = 0.9961 mol/L-min
r = 0.9876 mol/L-min
r = 0.9781 mol/L-min
r(max) = 0.4336 mol/L-min
km = 2.8096 mol/L
r = 0.9564 mol/L-min
r(max) = 0.1457 mol/L-min
km = 2.5083 mol/
Therefore, Langmuir isotherm best fit the data with r = 0.9968 for withoutinhibitor.
Substrate
mole/mL
Inhibitor
mole/mL
4.7
4.7
4.7
10.8
10.8
10.8
30.3
30.3
30.3
0
7.57
30.30
0
7.58
30.30
0
7.58
30.3
0.0434
0.0285
0.0133
0.0713
0.0512
0.0266
0.1111
0.0909
0.0581
Calculate (a) the value of Michaelis-Menten constants of the enzyme, Ks, and (b) the dissociation
constant of enzyme-inhibitor complex, KI.
600
500
400
300
200
100
0
10
a. @ I=0
Rmax = 1/m = 0.1569
Ks = bRmax = 12.5839
15
20
25
30
35
@ I = 30.30
Rmax = 1/m = 0.1560
KI = bRmax = 51.3368
@ I= 7.58
Rmax = 1/m = 0.1537
KI = bRmax = 21.0720
CHAPTER 2: ENZYME KINETICS ; ADDITIONAL PROBLEM
https://ww2.chemistry.gatech.edu/~lw26/bCourse_Information/3511/stud_comp/chap12_17.pdf
The following data were obtained for the reaction A B, catalyzed by the enzyme Aase. The
reaction volume was 1mL and the stock concentration of A was 5.0mM. Seven separate reactions
were examined, each containing a different amount of A. The reactions were initiated by adding
2.0L of a 10M solution of Aase. After 5 minutes, the amount of B was measured.
Reaction
1
2
3
4
5
6
7
Volume of A
added(L)
8
10
15
20
40
60
100
Amount of B present
at 5 minutes (nmoles)
26
29
39
43
56
62
71
(a) Calculate the initial velocity of each reaction (in units of M.min-1)
(b) Determine the KM and Vmax of Aase from a Lineweaver-Burk plot.
(c) Calculate kcat.
SOLN:
(a) o = (26nmol/5min) / (1.0mL) x (103 mL/L) x (.001 mol/1nmol) = 5.2 M.min-1
Reaction
1
2
3
4
5
6
7
o(M.min-1)
5.2
5.8
7.8
8.6
11.2
12.4
14.2
[S] M
(M.min-1)
1
2
3
4
5
6
7
40
50
75
100
200
300
500
0.025
0.02
0.0133
0.010
0.005
0.0033
0.002
5.2
5.8
7.8
8.6
11.2
12.4
14.2
(y) 1/ (min-1/
M)
0.192
0.172
0.128
0.0116
0.089
0.081
0.070
b)
CHAPTER 2: ENZYME
KINETICS ; ADDITIONAL PROBLEM
Pesticide inhibition on enzyme has been reported, which caused the enzyme activity to reduce.
The collected data with and without inhibition are presented below. Determine the type of
inhibition and the KI for the inhibitor.
[S], M
Rate [I=0],
M/min103
Rate
[I=20nM],
M/min103
3.3010-4
56
5.0010-4
71
6.7010-4
88
1.6510-3
124
2.2110-3
149
37
47
61
103
125
1 Km 1
1
=
+
V V max S V max
=5.003310-6
Km
-9
V max =4.320010
K m = 8.634310-4 M
K mapp
-9
V max =7.460510
K mapp = 1.443310-3 M
[ ]
K m 1+
I
=K mapp
; 8.634310-4 M
KI
K I = 2.978010-8 M
20 109 M
1+
= 1.443310-3 M
KI
SOLUTION:
K =A e
E a
RT
@T1=55 C:
0.33= A e
125 kJ /mol
8.314 kj
(55+273)K
kmol .K
A=2.60991019
@T2=100 C:
K100C = (2.60991019) e
K100C =82.8182/s
125kJ / mol
8.314 kj
(100+273)K
kmol .K
1.25
3.84
5.81
7.13
0.398
0.649
0.859
1.00
When 2.010-3 mol/dm3 phenyl butyrate ion was added to the solution, the results were:
CBGP mol
,
102 dm 3
Rate,
mol
dm3 . s
1.25
2.50
4.00
5.50
0.172
0.301
0.344
0.548
In a separate experiment, the effect of 5.010-2 mol/dm3 benzoate ion was monitored and the
results were:
CBGP mol
,
102 dm 3
Rate,
mol
dm3 . s
1.75
2.50
5.00
10.00
0.183
0.201
0.231
0.246
Determine the type of inhibition and KI for phenyl butyrate and benzoate ion.
SOLUTION:
Assuming Lineweaver-Burk Equation:
Line 1: without inhibition
1 Km 1
1
=
+
V V max S V max
y=0.8126+0.0216x
V max = 1.2306 mol/dm3.s
K m = 0.0266 mol/dm3
TYPE OF INHIBITION:
[ ]
K m 1+
KI
I
2.0 103
=K mapp
0.0266
1+
=0.0592
;
KI
KI
= 1.636910-3 mol/dm3
KI
V max
I
1+
KI
; 0.2665
= 0.0138 mol/dm3
1.2306
5.0 102
1+
KI