Beruflich Dokumente
Kultur Dokumente
ChE-5201
Agbay, Philip D.
1. For a given species of an enzyme that doubles every 3 hours, what is the mass of the
biomass that may be expected from 100 liters of seed if each liter contains 8 grams
biomass the first order removal rate constant is 2.6 per hour.
SOLUTION:
t = ln
3 hr = ln
k = 0.231
Using the obtained value of k = 0.231, and substituting to the new conditions
t = ln
24 = ln
C = 204 kg
(a) Determine the specific growth rate during the growth phase.
(b) What is the culture doubling time
SOLUTION:
(a) The data are plotted as in Figure E8.1.
= 0.67 d 1
SOLUTION:
V = Vmax
Km = 18.89
Vmax = K3 Eo
K3 = 35 / day
V = K 3 Eo
= 35 x 2 x
V = 20.8256 / day
Atienza, Angielle A.
ChE - 5201
cs / (mol dm-3)
0.062
1.82
4.96
5.48
Solution:
a.
is in the form of
where
and
5mg/L of a dimeric enzyme having a total molecular weight of 82,340 g/mol (and
two active sites). The molecular weight of A is 119 g/mol. If the V max is found to be
0.13g/Ls and Km = 0.043 g/L, find the turnover number.
Reference: www.cmbe.engr.uga.edu
(Turnover
number)
Calculate the percentage of the substrate converted after 10 hour from the
beginning of the reaction in case that the initial concentration of the substrate
was 0.8 mol/dm3.
Reference: www.old.vsht.cz
For
Austria, Babylyn C.
PROBLEM #1:
Relation between Reaction Velocity and Substrate Concentration: MichaelisMenten Equation
a) At what substrate concentration will an enzyme with of 30 s -1 and a of 0.005
show one-quarter of its maximum rate?
b) Determine the fraction of
concentrations:, , and .
Answers:
a) Since and , , we can substitute into the Michaelis-Menten equation to give
0.5
1.0
1.5
2.5
3.5
23.5
32.2
36.9
41.8
44.0
(mM/min)
( (min/mW))
0.5 (2.0)
1.0 (1.0)
1.5 (0.67)
2.5 (0.4)
3.5 (0.27)
23.5 (0.043)
32.2 (0.0321)
36.9 (0.027)
41.8 (0.024)
44.0 (0.023)
with 10 mg/mL
ibuprofen(mM/min)
( (min/mW))
16.67 (0.06)
25.25 (0.0396)
30.49 (0.0328)
37.04 (0.027)
38.91 (0.0257)
where , , and
Using the linear regression, the following values are obtained:
Determination of
An enzyme is discovered that catalyzes the chemical reaction
SAD
HAPPY
A team of motivated researchers set out to study the enzyme, which they call
happyase. They find that the for happyase is . They carry out several experiments.
When and , the reaction velocity, , is 9.6 Ms-1. Calculate for the substrate SAD.
Answer:
We know , , , and We want to solve for . Substituting the known values allows us
to solve for .
Reference:
CourseSmart International E-Book for Principles of Biochemistry
by David L. Nelson, Michael M. Cox
1. Sucrose (A) isolated from fruits, and its enzyme sucrase (E) flow through a
mixed flow reactor (V = 6 liter) to undergo an enzymatic reaction that will
eventually synthesize glucose via hydrolysis of sucrose. From the entering
and leaving concentrations and flow rate find a rate equation to represent the
hydrolytic action of sucrase on sucrose.
For
reactor,
CEO, mol/L
CAO, mol/L
CA, mol/L
v, L/h
0.02
0.2
0.04
0.01
0.3
0.15
Solution:
0.001
0.69
0.60
1.2
mixed flow
the Monod
C A = -CM + k(
y = mx + b and = V/v
CEO, mol/L
CAO, mol/L
CA, mol/L
v, L/h
= V/v, h
,
mol/L
0.02
0.2
0.04
0.01000
0.01
0.3
0.15
1.5
0.01500
0.001
0.69
0.60
1.2
0.03333
, b = -CM
, b = -CM in the
Graph
Runs
CEO,
mol/L
CAO,
mol/L
CA,
mol/L
= V/v,
h
400
10
200
20
Solution:
For batch flow reactor, the
Monod kinetics will be
represented by
= -CM + k(
y = mx + b and = V/v
Thus, linearizing the equation will let y =
=, m = k, x =
,b
= -CM
Runs
CEO, mol/L
CAO, mol/L
CA, mol/L
= V/v, h
400
10
0.81326
105.7232
200
0.54217
52.86158
20
0.33381
6.342356
=, m = k, x =
, b = -CM in
Graph:
cellulase
cellulose
sugar
and both celluboise and glucose act to inhibit the breakdown. To study the
kinetics of this reaction a number of runs are made in a mixed flow reactor kept
at 50C and using a feed of finely shredded cellulose (C AO =25 kg/m3), enzyme
(CEO = 0.01 kg/m3, same for all runs), and various inhibitors. The results are as
follows:
Runs
Exit streams,
CA, kg/m3
(No inhibitor)
,min
(w/cellobiose)
, min
(w/glucose)
, min
1.5
587
940
1020
4.5
279
387
433
9.0
171
213
250
21.0
36
40
50
, b = -CM
(1+NCBo)
Runs
Exit
streams, CA,
kg/m3
(No
inhibitor)
,min
(w/cellobiose)
, min
1.5
587
940
0.3747
0.6000
4.5
279
387
0.6124
0.8495
9.0
171
213
0.9619
1.1981
21.0
36
40
1.8900
2.1000
For no inhibition:
Using linearization and inputting y = CA, m = k, x =
calculator, one can get
m = k = 12.844/min
-b = CM = 3.365 kg/m3
, b = -CM in the
, b = -CM
Then the Michaelis Menten equation for competitive inhibition will become,
r=
The final rate equation based from Michaelis-Menten for competitive inhibition
equation is r =
Graph:
Reference:
Chemical Reaction Engineering Third Edition, Octave Levenspiel
Cantos, Jonathan C.
V0 (mol/L-min)
0.1
0.27
2.0
5.0
10.0
20
20.0
40
40.0
64
60.0
80
100.0
100
200.0
120
1000.0
150
2000.0
155
SOLUTION:
We can use the Lineweaver-Burk equation for the analysis. For this, the
reciprocals of the entries in Table 1 must be calculated and then plotted as shown in
Fig. 1.
(a) From the reciprocal of the ordinate intercept, Vmax = 160 mol/L-min, and
(b) From the reciprocal of the abscissal intercept, Km = 60 mol/L .
2. Use the Michaelis-Menten equation to complete the enzyme kinetic data set, when
Km is known to have a value of 1 mmol/L.
[S]0 (mol/L)
0.5
V0 (mol/L-min)
50
1.0
2.0
3.0
10.0
__
__
__
__
SOLUTION:
Using the Michaelis Menten equation, the first entry in the table gives Vmax = 150
mol/L-min. The other entries simply follow by substituting the values of [S] 0 into
V0 = . The results are as follows:
[S]0 (mol/L)
0.5
1.0
2.0
3.0
10.0
V0 (mol/L-min)
50
75
100
112.5
136.4
SOLUTION:
(a) For glucose the values of V0/Vmax are 0.5, 0.91, and 0.99, respectively; for fructose
the values are 0.091, 0.56, and 0.91.
(b) Glucose; at lower concentrations the reaction rate is a greater fraction of V max than it
is with fructose.
Dimayuga, Evytte M.
1. From the following kinetic data, estimate Vmax and Km for the catalysis of
Substrate 1 by the enzyme Questionase. Draw a curve for the data you would
expect to observe for Substrate 2 if Km = 30 uM for this substrate (assuming the
both have the same Vmax).
Solution:
Determining Vmax for Substrate 1 might vary, depending on how you draw the
curve. The value of Vmax = 200 uM/sec) was used to calculate values for the data.
Whatever you chose, Km will be determined from the substrate concentration that gives
you Vmax/2. Your curve for Substrate 2 might look a little different as well.
2. If [Questionase] = 0.05 uM, calculate kcat for Substrate 1 and what would be the
value for kcat/Km in units of M-1s-1? (Hint: you need to convert Km to units of
Molar)
Solution
Vmax = kcat [Etotal]
kcat
= Vmax / [Questionase]
= (200 uM/sec)/0.05 uM
= 4000 s-1
Km
= 10 uM
= 10 x 10-6 M
= 1 x 10-5 M
respectively. Determine the effectiveness factor and the initial reaction rate, when
the substrate concentration is 0.6 kmolm_3.
Solution
The value of the Thiele modulus is calculated from Equation 7.21.
de Guzman, Monroe H.
PROBLEM NO. 1 THE FASTEST ENZYME
The reaction rate of carbonic anhydrase is one of the fastest of all
enzymes, and its rate is typically limited by the diffusion rate of its substrates
(Badger, 1994). The table below shows the data of the enzyme with respect to its
rate of reaction in a specific type of substrate.
Table 1.0 Carbonic anhydrase kinetics
S (mmol/L)
0.10
0.15
0.20
0.25
0.30
0.35
0.40
-rs (mmol/Ls)
1.2 x 106
1.7 x 106
2.1 x 106
2.4 x 106
2.9 x 106
3.2 x 106
3.6 x 106
S (mmol/L)
0.45
0.50
0.55
0.60
0.65
0.70
0.75
-rs (mmol/Ls)
4.0 x 106
4.6 x 106
5.2 x 106
6.0 x 106
6.9 x 106
7.5 x 106
8.2 x 106
Solution:
To solve this problem, the Michaelis-Menten constant (K m), limiting or
maximum velocity (Vmax), and the substrate concentration (S) must be
determined.
Solution:
To determine the amount of the competitive inhibitor, the data given in
problem number 1 must be utilized prior to the fact that it is accurate in dealing
with the kinetics. The obtained constants will be used to solve for the
concentration of the said inhibitor.
Using the obtained constants: Km = 1.63816 mmol/L and Vmax = 2.0096463
7
x 10 mmol/L-s, and on the basis of 1 L solution of enzyme:
S = 0.172/100 = 1.72 x 10-4 mol/L
S = 0.172 mmol/L
The equation for competitive inhibition is given by:
v = Vmax[S]/{[S] + Km(1 + [I]/KI)}
1.13 x 104 = 2.0096463 x 107[0.172]/{[0.172] + 1.63816(1 + [I]/2.1388)}
[I] = 397.01404 mmol/L
Thus the students added 0.39701 M strychnine.
After determining the concentration, the professor was impressed and got
the idea of determining an aid in HIV. To accomplish this, he needs the constant
of inhibition provided by the enzyme peptide-based HIV-1 protease. He has done
researches about the inhibitor.
1. HIV-1 protease is an organic compound exhibiting uncompetitive
inhibition to carbonic anhydrase.
2. Its aqueous solution lowers the freezing point of water to -0.0771C.
3. It forms an enzyme-substrate complex with the carbonic anhydrase that
follows Lineweaver-Burke plot.
4. The inhibitor affects the speed of an enzyme by lowering the speed
three-folds.
As compensation to what the students did, the whole batch was assigned
to rework the enzyme and determine the constant of inhibition (K I) of HIV-1
protease for the professor to formulate the medicine. The aqueous enzyme is
preserved inside the refrigerator with the freezing point lowered by 0.0122 K.
To determine the concentration of the substrate and the inhibitor:
T = Kfmi
For HIV-1 protease:
0 + 0.0771 = 1.86(m)(1)
m = 0.04145 m ~ 0.014145 mol/L
[I] = 41.45161 mmol/L
For carbonic anhydrase:
0.0122 = 1.86(m)(1)
m = 6.5591 x 10-3 m ~ 6.5591 x 10-3 mol/L
[S] = 6.55914 mmol/L
At [S] = 6.55914 mmol/L, evaluate the speed (v).
v = Vmax[S]/(Km + [S])
v = 2.0096463 x 107(6.55914)/(1.63816 + 6.55914)
v = 1.60804 x 107 mmol/L-s
BS ChE
5201
Enzyme Kinetics
Given:
For 1g of bacteria
Vmax=45g/day
V=28g/day
S=30mg/L
V=Vmax *S/(Km+S)
28g/day=[(45g/day)((30mg/L)/(1g/1000mg))]/(Km+0.03g/L)
Km=0.0182g/L
V=54.9618g/day
2.It is desired to reduce the bacterial count of polluted water from 40 million
organisms per mL to 9 organisms per mL. Calculate the number of
completely mixed chlorine contact chambers in seies, each having a
detention time of 3 hours that would be required if the first order removal
rate constant is 3.6/hr
Given:
Ca=9organisms per mL
Cao=40000000organisms per mL
K=3.6/hr
T=3 hr
Dimaano, Jezza B.
Problem #1.
With the following enzyme activity results determine Vmax.
[S] (mol/L)
2 x 10-1
2 x 10 -2
2 x 10 -3
2 x 10 -4
1.5 x 10 -4
1.3 x 10 -5
V (mol/min)
60.00
60.00
60.00
48.00
45.00
12.00
Answer:
From the available data, we can notice that the reaction rate doesnt
increase when the substrate concentration is over 2 x 10-3 M. This is the
maximal velocity (Vmax) of the enzyme, in this case 60 mol/min.
Problem #2.
The results for enzyme activity analysis can be found below. Determine :
a) Vmax;
b) Km;
[S] (mol/L)
5 x 10-2
5 x 10 -3
5 x 10 -4
5 x 10-5
5 x 10-6
5 x 10 -7
v (mol/min)
0.25
0.25
0.25
0.20
0.071
0.01
Answer:
a) Vmax = 0.25 mol/min;
b) Km can be determined using the Michaelis-Menten equation:
v = [S] Vmax
[S] + Km
v[S] + vKm = [S]Vmax
vKm = [S]Vmax - v[S]
vKm = [S] (Vmax - v)
Km = [S] (Vmax - v)/v
v (mol/min)
65.00
63.00
51.0
33.00
27.00
17.00
[S] + Km
v[S] + vKm = [S]Vmax
vKm = [S]Vmax - v[S]
vKm = [S] (Vmax - v)
Km = [S] (Vmax - v)/v
Using the data for a [S] of 1 x 10-6:
Km =
Km = 2.8235 X 10-6 M
b) Vmax = 65.00 mol/min
Em
(mM)
Vmax
(mMol/sec)
4.0
25
0.5
15
Which substrate will react most rapidly at low substrate concentration (<< Km)?
Solution:
Since at low substrate concentration [S], Vmax = kcat [Et]. As we have
Vmax, which is directly proportional to kcat at a given enzyme concentration, so
comparison of Vmax/Km gives idea about enzyme's efficiency with substrates.
The value of substrate B = Vmax/Km = 30 (mol/sec/mM) and for A = 6.25
(mol/sec/mM). Hence at low concentration substrate B will be used more
efficiently to react compared to substrate A.
2. The following table shows the rate of reaction of substrate to product in the
presence of enzyme; v (mol/sec) under different conditions;
1. Reaction 1: Reaction of substrate with enzyme to form product in the
absence of inhibitor.
2. Reactions 2 and 3: Reaction takes place in the presence of two different
inhibitors, each with 10mM concentration.
[S] mM
v, reaction 1
( mol/sec)
v, reaction 2
(inhibitor A)
( mol/sec)
v, reaction 3
(inhibitor B)
( mol/sec)
2.5
1.17
0.77
4.0
2.1
1.25
6.3
4.0
2.0
10
7.6
5.7
2.5
20
9.0
7.2
2.86
Lets take same amount of enzyme in each case and determine K m and Vmax for
the enzyme (in absence of inhibitors) and determine K iand the type of inhibition
for each inhibitor.
Solution:
Km = 3.3 mM, Vmax = 10 mmol/sec
1. Inhibitor A (competitive inhibition), there will be no change in V max , but
Kmapp = Km{1 + ([I]/KI)}-1/Kmapp = -0.13mM-1 Kmapp = 7.7mM K mapp /Km =
7.7mM/3.3mM= 2.33 = 1 +[I]/ KI = 1+ [10mM]/ KI KI (inhibitor-A) =10mM/
2.33 1 = 10mM/ 1.33 = 7.52 mM
2. Inhibitor B (pure non-competitive) there will be no change in K m; but
Vmaxapp = Vmax / {1 + ([I]/ KI )} 1/ Vmaxapp = 0.3( m o l / s e c )-1 Vmaxapp =
3.33 mo l / s e c Vmax/ Vmaxapp = 10 mol/sec/ 3.33 m o l / s e c = 3 = 1 +
[I]/KI = 1 +[10mM]/ KI KI (inhibitorB) =10mM / 3 - 1 = 10mM/2 = 5.0 mM
3. Draw a Lineweaver-Burk plots for an enzyme for which the following data are
available.
[S]
(mM)
(mmol min-1)
(mmole min-1)
3.0
4.58
3.66
5.0
6.40
5.12
7.0
7.72
6.18
9.0
8.72
6.98
11.0
9.50
7.60
What are the Km and Vmax values for the inhibited and uninhibited enzymes? Is the
inhibitor competitive or non-competitive?
Solution:
First determine ; ; then plot
Plot 1: vs
Plot 2: vs
0.3333
0.2000
0.1429
0.1111
0.0909
0.2183
0.1562
0.1295
0.1147
0.1053
For ; x = & y =
y = 0.4665x + 0.0629
x = 0 ; y = 0.0629
Vmax (no I) = 1/0.0629 = 15.8983 mmol min-1
For ; x = & y =
y = 0.5846x + 0.0784
x = 0 ; y = 0.0784
Vmax (I) = 1/0.0784 = 12.7551 mmol min-1
Non - competitive inhibition
0.2732
0.1953
0.1618
0.1433
0.1316
Francisco, Jayvee D.
1. The following mechanism relates to an enzyme E with two binding sites for
the substrate S. Two complexes are formed: a reactive binary complex ES,
and a nonreactive ternary complex ESS.
? At
Solving for cE and cESS in terms of cES and cS from (1) and (2), respectively, and
substituting the results in (3) and rearranging to obtain cES in terms of cEo and cS,
we have
where Km is the Michaelis constant (equation 10.2-17). Substituting the result for
cES from (5) into (4) we obtain the rate law:
2. The hydrolysis of sucrose (S) catalyzed by the enzyme invertase has been
studied by measuring the initial rate, rPo, at a series of initial concentrations of
sucrose (cSo). At a particular temperature and enzyme concentration, the
following results were obtained (Chase et al., 1962):
Determine the values of Vmax and the Michaelis constant Km in the MichaelisMenten rate law.
SOLUTION:
Linear regression of the data given, according to equation 10.3-2, results in Vmax
= 0.46 mol L-1 s-1, and Km = 0.043 mol L-1. Figure 10.2 shows the given
experimental data plotted according to equation 10.3-2. The straight line is that
from the linear regression; the intercept at l/cSo = 0 is l/Vmax= 2.17 mol-l L s, and
the slope is Km/Vmax = 0.093 s.
SOLUTION:
The results given in Table 3.3 are plotted as shown in Figure 3.9. This
LineweaverBurk plot shows that the mechanism is competitive inhibition. From
the line for the data without the inhibitor, K m and Vmax are obtained as 0.98 gmol
m-3 and 9.1 mmol m-3 s-1, respectively. From the slopes of the lines, K I is
evaluated as 0.6 gmol m-3.
Macalintal, Cristel M.
Carbonic anhydrase catalyzes the hydration of CO 2.
CO2 + H2O H2CO3
The Km of carbonic anhydrase for CO2 is 12 mM. Carbonic anhydrase gave an
initial velocity vo=4.5 mols H2CO3 formed/mL sec when [CO2]=36 mM. What is
the Vmax for this enzyme?
Solution:
Given:
For an enzyme that displays Michaelis- Menten kinetics, what is the reaction
velocity, V (as a percentage of Vmax), observed at:
a. [S]=Km
b. [S]=0.5Km
Solution:
a.
b.
Magboo, Michael S.
1. A 10 m3 chemostat operating at 75% capacity is producing biomass from a
glucose feed at a volumetric rate of 46.9 L/min. The specific growth rate of the
enzyme used is
Q = () () () () = 0.5 m3 /hr
2. The hydrolysis of sucrose (S) catalyzed by the enzyme invertase has been
studied by mea- surmg the initial rate, rpo, at a series of initial concentrations of
sucrose (cS0). At a particular temperature and enzyme concentration, the
following results were obtained (Chase et al., 1962):
cs,/mol L-l
0.0292
0.0584
0.0876
0.117
0.146
0.175
0.234
0.265
0.311
0.330
0.349
0.372
Determine the values of Vmax and the Michaelis constant Km in the MichaelisMenten rate law.
SOLUTION:
Linear regression of the data given, results in Vmax = 0.46 mol L-l s-l, and
Km = 0.043 mol L-l. Figure 10.2 shows the given experimental data plotted
according to equation 10.3-2. The straight line is that from the linear regression;
the intercept at l/cSo = 0 is l/Vmax = 2.17 mol-l L s, and the slope is Km/Vmax =
0.093 s.
3. The following mechanism relates to an enzyme E with two binding sites for the
substrate S. Two complexes are formed: a reactive binary complex ES, and a
nonreactive ternary complex ESS .
We apply the SSH to the complexes ES and ESS; in the latter case, this is
equivalent to assumption of equilibrium with the dissociation constant for ESS
given by K2 = k-,/k,:
Solving for cE and cESS in terms of cEs and cs from (1) and (2), respectively, and
substituting the results in (3) and rearranging to obtain c ES in terms of cEO and cs,
we have
where Km is the Michaelis constant (equation 10.2-17). Substituting the result for
cus from (5) into (4) we obtain the rate law:
[S] (mol/L)
0.25
0.25
0.25
0.20
0.071
0.0096
v (mol/min)
0.25
0.25
0.25
0.20
0.071
0.0096
Matibag, Cherriellyn B.
1) For an enzyme (5 M) , the following initial velocities have been reported
depending on the substrate concentration:
[Substrate] mM
0.02
0.04
0.07
0.1
0.15
0.2
0.3
0.5
0.7
Draw a Michaelis-Menten plot for this enzyme.
V0,mM, s-1
10.83
18.57
26.76
32.50
39.00
43.33
48.75
54.17
56.88
2. You are trying to reproduce experimental data from the previous student in the
lab. He/she had reported that the enzyme under investigation has a kcat of 1875
s-1 and a catalytic efficiency of 7.5 107 M-1 s-1. What is the KM of this enzyme?
From kcat/KM and kcat, you can calculate KM as
Solution:
Km =
3. In repeating the measurement, you want to have a vmax of 10 mM s-1 (as this
is easy to measure). How much enzyme should you use? Provide your result in
M.
Solution:
Bernadeth R. Pagcaliwagan
1. Exploring the Michaelis-Menten equation I. What is the v/Vmax ratio when [S]
= 4Km?
2. Given a specie of a microorganism that doubles every 2.5 hours, the mass of
biomass that may be expected from 500 liters of seed if each liter contains 5
grams biomass and the fermentation culture was maintained for 12 hours.
a. Note that the original protein solution has been diluted by a factor of 10 as a
result of the assay, and there are 1000 mol in 1 mmol.
b.
and
or
Acetyl cholinesterase
acetylcholine:
catalyzes
the
hydrolysis
of
the
neurotransmitter
, both of which is
Rivera, Lourelane H.
1. The concentration-velocity data shown below were obtained for an
enzyme catalyzing a reaction S
P. Calculate Km and Vmax.
(S)
V
M
nmoles x liter -1 x min-1
2.5 x 10-6
24
3.33 x 10-6
30
-6
4.0 x 10
34
5 x 10-6
40
-5
1 x 10
60
2 x 10-5
80
4 x 10-5
96
-4
1 x 10
109
2 x 10-3
119
-2
1 x 10
120
Linearizing the Michaelis-Menten equation:
Solution:
Let x = ; y =
By linear regression using the calculator:
y-intercept = A = = 8343317.435
Therefore,
M/min
Slope = B = = 83.4440
Therefore,
/min
2. Estimate k, the first-order rate constant, for an enzyme preparation with a
Vmax of 4.6 under the given experimental conditions. M.
Solution:
3. The velocity of an enzyme-catalyzed reaction was measured at several
substrate concentrations. Calculate Km and Vmax for the reaction.
[S]
(M)
0.25
0.5
1.0
2.0
4.0
V
(mM/sec)
0.75
1.20
1.71
2.18
2.53
Solution:
By linearizing Michaelis-Menten equation:
Let x = ; y =
By linear regression using the calculator:
y-intercept = A = = 333.6629
Therefore,
M/sec
Slope = B = =
Therefore,
/sec