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SM ch3 Bio Shuler 3
SM ch3 Bio Shuler 3
SM ch3 Bio Shuler 3
Problem 3.1
rate k5 ES 2
r1 S E r2 ES 1
(1)
r3 ES 1 r4 ES 2
(2)
E 0 E ES 1 ES 2 (3)
r
ES 1 4 ES 2
from (2) r3 (4)
r2 r4 ES 2
E
r1r3 S
from (1) + (4) (5)
r2 r4 r1 r4 S r1 r3 S
E 0 ES 2
r1 r3 S
r1 r3 E 0 S
ES 2
r2 r4 r1 r4 S r1 r3 S
r5 E 0 S r2 r
rate where Km1 , Km 2 4
Km 2 Km1 S S r1 r3
d ES 1
r1 E S r2 ES 1 r4 ES 2 r3 ES 1 0
dt (6)
d ES 2
r3 ES 1 r4 ES 2 r5 ES 2 0
dt (7)
r4 r5
ES 1 ES 2
from (7) r3 (8)
r2 r4 r2 r5 r3 r5
E ES 2
from (6) + (8): r1 r3 S (9)
1
r2 r4 r2 r5 r3 r5 r1 r4 S r1 r5 S r1 r3 S
E 0 ES 2
r1 r3 S
r1 r3 E 0 S
ES 2
r2 r4 r2 r5 r3 r5 r1 r4 S r1 r5 S r1 r3 S
r5 E 0 S
rate
Km2 r5 r3 Km1 S r5 r1 S
r r
Km1 2 Km 2 4
where r1 and r3
Problem 3.2
* r values are the same as k values all are rate constants
E S 1 ES 2 E P
r r
r1 r2
d ES
r1 E S r2 E P r1 ES r2 ES 0
dt (1)
d P
rate r2 ES r2 E P
dt (2)
E 0 E ES , E E 0 ES
(3)
r1 r2
E ES
r1 S r2 P
from (1): (4)
r1 S r2 P r1 r2
E 0 ES
r1 S r2 P
Combine (3) + (4):
E 0 r1 S r2 P
ES
r1 S r2 P r1 r2
(5)
r1 r2 E 0 S r2 r2 E 0 P
rate r 2 E 0 ES P
r1 r2 r1 S r2 P
r1 r2 E S r1 r2 E 0 P
rate
r1 r2 r1 S r2 P
2
r1 Vs S r2 VP P
rate
r1 r2 r1 S r2 P
rate
V s K1m S VP K P P
S P
1 1
K m KP
Problem 3.3
k 1 k 2
Km 4.5 105 M
a) k1
E 0 106 M
S 103 M
V S
V m1
b)
Km S
V rE
but m 2 0
V 9.58 104 M sec 1
Problem 3.4.
Problem 3.5.
3
And from the intercept on X-axis, - 1/Km = -1.2 and Km = 0.83 mM
From 1/V versus 1/S plot for I = 1.3 mM and S0 = 0.50 mM V = 1.3 mM/h
Then, V = Vm S/ (Km(1+I/Ksı)) + S , 1.3 = 5(0.5)/ (0.83(1+1.3/Kı) + 0.5 )
Then Kı = 1.82 mM
Problem 3.6 .
Problem 3.7.
Problem 3.8
E
H 2 O CO 2 HCO3 H
Plot 1/V versus 1/S (Lineweaver – Burk plot)
1 1 1
y – intercept = Vm k 2 E 0 , x – intercept = K m
1 40
4 103
V Co 2
Hydration:
1 163.15
1.31 104
V HCO3
Dehydration:
4
1 V 7.61105 M sec 1
1.31 104 M 1 sec m
Vm r2 2.72 104 sec 1
1 1.31104 1
M K m 1.24 102 M
Km 163.15
Problem 3.9
1 1
9.525 0.60
a.) Plot 1/V versus 1/S: V S
1 0.60
0.063M 1 K m,app 16.0 g S / L
K m,app 9.525
SinceKm,app> Km the inhibitor is competitive.
b.)
5
I
K m,app K m 1
KI
KI
I 1.37g I L
K m,app K m 1
Problem 3.10
1 mmol
Vm 3.31
b.) 0.302 ml.min
Problem 3.11
r
ATP
ATPase
ADP Pi or E S r 1 ES
r2
E P
1
6
But
S K m , V Vm r2 E
dp
r2 E 0e rt
dt
0.002
0
dp r2 E 0 e rt dt
0
r2 E 0
0.002 M 1 , E 0 2.0 10 8 M
r
mol g 6 g
2.0 108 1103 L 5 10 4 110 1.0g
L mol g
1.0 g
0.1
Fraction of pure enzyme 10 g
Problem 3.12
Vm1 Ss
V rL Sb Ss J
S
2
K m Ss s
K SI
Solving graphically,
a.) For [Sb] when the enzyme is inhibited and kL has an intermediate value, multiple steady
states are possible.
Multiple steady – states occur as the enzyme changes from being reaction controlled to
being diffusionally controlled.
7
b.) Yes. Diffusional limitations can decrease the substrate concentration such that it is no
longer inhibitory. Thus the apparent reaction rate will be greater than the intrinsic reaction
rate for [SS] less than [Sb].
Problem 3.13
Plotting the date and using the data points for high substrate concentration,
Vm = 130 μmol/min, Km = 8.24 μmol/L
From the plot it is obvious that the data do not fit into Michaelis – Menten kinetics at low
substrate concentrations.
Some of the error may be attributed to the method since both axes contain v. However, there
are two possible explanations for the deviations from Michaelis – Menten Kinetics:
Problem 3.14
8
Obtain rates by taking tangents at specific time points.
d P
v slope of tangent
dt
Obtain the amount of substrate present for both cases by a mass balance.
H 2O Starch
dextrose
MW 18 180
Using initial time data, a plot of 1/V versus 1/S can be made.
9
Soluble:
1
0.16
Vm
1 mg 1
Vm
0.16 mL min 11600 units
mg
5.39 104
mL min unit enzyme
1 mg
Km 575
0.00174 mL
Immobilized:
1 1 mg 1 4 mg
0.12, Vm 1.80 10
Vm 0.12 mL min 46 400 units mL min unit of enzyme
1
Km 575 mg mL
0.00174
Vm r2 E 0 E 0 Ae Ea RT
Ea Ea
ln Vm ln E 0 ln A slope
RT R
cal Kcal
E a 8299K 1.987 16.5
mol K mol
10
Immobilized: lnVm = 35.65 – 6582 (1/T)
cal Kcal
E a 6582 K 1.987 13.1
mol K mol
Problem 3.15
In vitro batch reactors represent a closed system of constant volume, thus Michaelis-Menten
Kinetics and the quasi-steady-state approximation will not describe the system when E0 ≈ S0.
However, intracellular enzyme reactions are open systems where there is a continuous supply
and depletion of substrate and product provided by the interaction of cellular compartments
and the intracellular and extracellular environments. E0 and S0 may be the same, but the
concentrations may be orders of magnitude different either in different organelles or inside
versus outside the cell.
Problem 3.16
Harry’s reasoning is wrong. The soluble enzyme is reaction controlled while the immobilized
enzyme may experience diffusional limitations. Thus the substrate is consumed more slowly
giving rise to a larger apparent half-life. The large particle size may result in an unused or
“reserved” catalytic capacity.
Problem 3.17
11
(b) Vm andK'm are determined from low substrate concentration where inhibition is not
ineffect:
1 Vm is at x 0 1 Vm 0.049333
Vm 20.3 mg l h
'
Km
E S
ES
r2
EP
S
K SI
ES2
Vm S
u
S
2
K S
'
m
K SI
du
0
d S
Determine KSI from maximum reaction rate; which is determined by setting
S max K 'm K SI
S max 90 mg 1 90 mg l 31.1 mg 1 K SI
K SI 260.5 mg 1
Km
1
From plot, S
12
(c) Rate of reaction at S = 70 mg/l?
Since inhibition effect is observed at this substrate, we must use (eg) 3.34
Vm S 20.3 70
u
S 70
2 2
K 'm S 31.1 70
K SI 260.5
u 11.9 mg l hr
Problem 3.18
We are given the reaction rate with diffusion limitation. To determine the reaction rate with
no diffusion limitation, lets look at what happens to the rate as the particle gets smaller, and
diffusional limitations are minimized. Aplot of Rate vs. particle size indicates that the rate
reaches a maximum at small particle size (0.1cm). At this size there is no diffusion limitation,
and the rate is N 200 mg/l hr
13
b. Assume negligible film resistance, so Sbulk = Ssurface Determine rate u/o diffusional
limitation from effectiveness factor:
rate at D P 0.5cm
rate w o diff
at D P 0.5cm
1/ Vm is at x 0 : y 0.0084626 Vm 118.2 mg uA / l h
–1/Km is at y = 0: 0 =0.0084626 + 0.41489 (–1/Km)
0.0084626 1
0.41489 Km
K m 49.0 mg uA l
Problem 3.19 a
Vm S0
rS
K S S0 (1)
Vm
R
K S De
(2)
3 (3)
14
Vm 0.5
10 ; Vm 14
From Equ 1. 0.2 0.5 (1)
Vm
0.1 1.04 Vm
0.2 1.5 105 3600
From Equ 2. (2)
Vm 14.55 Vm
Multiply (1) × (2) =
since 3 Vm 23.5 mmoles L h
14
0.595 0.6
23.5
3 0.595 5.04
b.
Vm
similarly, Vm 14; 0.2
0.2 1.5 105 3600
Vm 26.95 Vm 1.925 Vm
Problem 3.20
(a) Plot 1/V vs 1/S for all cases to determine the type of inhibition
15
Vmax is the same, regardless of inhibition, but as I increases, –1/Km decreases (apparent), that
is an increased value of Km,app, resulting in a reduction in reaction rate. This is characteristic
of Competitive Inhibition
Vm, Km can be determined from the I = 0 case.@ I = 0 (1/V) = 0.15782 + 0.19404 (1/S)
1 Vm at 1 S 0 : 1 Vm 0.15782
Vm 6.34 mM h
16
I 1.95 mM : 1 V 0.15343 0.40383 1 S
0 0.15343 0.40383 1 K m,app
0.15343 1
K m,app 2.63mM
0.40383 K m,app
I
K m,app K m 1
KI
1.26mM
I 1.26 mM : 1.95 mM 1.23mM 1
KI
1.26
1.58 1
KI
1.26
0.58 K I 2.16 mM
KI
1.95mM
I 1.95 mM : 2.63mM 1.23mM 1
KI
1.95
2.14 1 K I 1.71mM
KI
We would expect that KI is the same at different concentrations of inhibitor. The differences
in the two values shown here are due to experimental error.
Problem 3.21
K S 200 mg L 0.2 mg cm 3
VmSS
K L Sb S3
K m SS
K L 2.10 3 m sec 0.2 cm sec
Sb 1000 mg L 1mg cm 3
Then
0.1 SS
0.2 1 SS
0.2 SS
SS2 0.3SS 0.2 0
SS 0.62 mg cm3 620 mg L
b.
17
Vm SS
V K L Sb SS
K S SS
0.1 0.62
V 0.2 1 0.62 0.076 mg cm 2 sec.
0.2 0.62
Problem 3.22
Given
V J K L SB SS ; V K LSB K LSS ; y mx b
use
where
y V; K L SB b; SS X and K L slope
S1 20 mg L V 3.2 10 6 mg cm 2 s
b)
.005mg
S2 rate of supply rate of diffusion away V K L S2 surface
cm3
K m 5e 3 mg cm 3 ; Vm 4e 6 mg km 2 s
Vm S2 surface
3.2 106 mg cm 2 s 4e 5 cm s S2 surface 5e 3 mg cm 3
K m S2 surface
S2 surface .013mg cm3 13mg L
dP
2.89 106 mg cm 2 s
dt
Problem 3.23 a
18
Make total reaction rate curve →Vtot = 2.2.10 – 5 mg/cm2 S
2
1.25 105 mg cm S
P2 P1 1.25
c) 1.0 105 mg cm 2 S ratio of production rates
19