d. What type of inhibition is exhibited against NAD* as a cofactor? Describe what is going on in this type of inhibition. e. Referring to the mechanism below, what step is pyrazole likely interfering with given your answers in c and d? 1. binding of NAD+ to the enzyme, LADH 2. binding of C₂H5OH to LADH 3. Oxidation of C₂H5OH (This is the reaction step) and product dissociation 4. Dissociation of NADH from LADH NAD+ + LADH NAD LADH + NAD C₂H5OH LADH C₂H5OH NADH LADH + NAD LADH k₂ k + NAD LADH C₂H5OH NADH LADH + NADH+ LADH CH3CHO+H*
d. What type of inhibition is exhibited against NAD* as a cofactor? Describe what is going on in this type of inhibition. e. Referring to the mechanism below, what step is pyrazole likely interfering with given your answers in c and d? 1. binding of NAD+ to the enzyme, LADH 2. binding of C₂H5OH to LADH 3. Oxidation of C₂H5OH (This is the reaction step) and product dissociation 4. Dissociation of NADH from LADH NAD+ + LADH NAD LADH + NAD C₂H5OH LADH C₂H5OH NADH LADH + NAD LADH k₂ k + NAD LADH C₂H5OH NADH LADH + NADH+ LADH CH3CHO+H*
Biochemistry
6th Edition
ISBN:9781305577206
Author:Reginald H. Garrett, Charles M. Grisham
Publisher:Reginald H. Garrett, Charles M. Grisham
Chapter27: Metabolic Integration And Organ Specialization
Section: Chapter Questions
Problem 4P
Related questions
Question
Answer for D, and E. The answers for A, B, and C are down below.
![3. Pyrozole has been proposed as a possible nontoxic inhibitor of LADH-catalyzed ethanol oxidation. Its
kinetics have been studied and, in 2 separate experiments, the velocity of the reaction was measured as a
function of [C₂H5OH] and of [NAD+]. The data is given in the tables below.
[C₂H5OH] varies, [LADH]= 4µg/ml, [NAD+] = 350 µM, pH=7.4, 23.5C
[C₂H5OH] Vo (relative
units)
Vo (relative units)
1x10-5 M pyrazole
M
No pyrazole
2.1
1.8
0.008
0.002
0.001
0.00067
1.5
1.35
Vo (relative
units)
No pyrazole
0.00011
1.7
0.000056
1.4
0.000033
1.3
0..000017 0.95
1.7
1.0
[NAD+] varies, [LADH]= 4µg/ml, [C₂H5OH]= 5 mM, pH=7.4, 23.5C
[NAD+]
Vo (relative units)
4x10-5 M pyrazole
0.69
0.52
0.85
0.71
0.63
0.47
a. Plot the data as Lineweaver-Burke plots on two separate graphs.
b. Determine the Km values for [C₂H5OH] and of [NAD*]. (You can't find Vmax because the exact units of Vo
are not provided)
c. What type of inhibition is exhibited by pyrazole against C₂H5OH is a substrate? Describe what is going on in
this type of inhibition.
2. binding of C₂H5OH to LADH
d. What type of inhibition is exhibited against NAD+ as a cofactor? Describe what is going on in this type of
inhibition.
e. Referring to the mechanism below, what step is pyrazole likely interfering with given your answers in c and
d?
1. binding of NAD+ to the enzyme, LADH
3. Oxidation of C₂H5OH (This is the reaction
step) and product dissociation
4. Dissociation of NADH from LADH
NAD + LADH = NAD LADH
k_1
NAD LADH + C₂H5OH
NAD
LADH C₂H5OH
NADH LADH
k2
2
NAD LADH C₂H5OH
ОН
NADH LADH +
NADH+LADH
CH3CHO+H*](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fd2705ec0-2034-4d28-a385-06c28bb62d2f%2F017e1983-11c6-46c7-ab0d-29d67e34de55%2Fynym9x5_processed.png&w=3840&q=75)
Transcribed Image Text:3. Pyrozole has been proposed as a possible nontoxic inhibitor of LADH-catalyzed ethanol oxidation. Its
kinetics have been studied and, in 2 separate experiments, the velocity of the reaction was measured as a
function of [C₂H5OH] and of [NAD+]. The data is given in the tables below.
[C₂H5OH] varies, [LADH]= 4µg/ml, [NAD+] = 350 µM, pH=7.4, 23.5C
[C₂H5OH] Vo (relative
units)
Vo (relative units)
1x10-5 M pyrazole
M
No pyrazole
2.1
1.8
0.008
0.002
0.001
0.00067
1.5
1.35
Vo (relative
units)
No pyrazole
0.00011
1.7
0.000056
1.4
0.000033
1.3
0..000017 0.95
1.7
1.0
[NAD+] varies, [LADH]= 4µg/ml, [C₂H5OH]= 5 mM, pH=7.4, 23.5C
[NAD+]
Vo (relative units)
4x10-5 M pyrazole
0.69
0.52
0.85
0.71
0.63
0.47
a. Plot the data as Lineweaver-Burke plots on two separate graphs.
b. Determine the Km values for [C₂H5OH] and of [NAD*]. (You can't find Vmax because the exact units of Vo
are not provided)
c. What type of inhibition is exhibited by pyrazole against C₂H5OH is a substrate? Describe what is going on in
this type of inhibition.
2. binding of C₂H5OH to LADH
d. What type of inhibition is exhibited against NAD+ as a cofactor? Describe what is going on in this type of
inhibition.
e. Referring to the mechanism below, what step is pyrazole likely interfering with given your answers in c and
d?
1. binding of NAD+ to the enzyme, LADH
3. Oxidation of C₂H5OH (This is the reaction
step) and product dissociation
4. Dissociation of NADH from LADH
NAD + LADH = NAD LADH
k_1
NAD LADH + C₂H5OH
NAD
LADH C₂H5OH
NADH LADH
k2
2
NAD LADH C₂H5OH
ОН
NADH LADH +
NADH+LADH
CH3CHO+H*
![Step 1
Since you have posted a question with multiple sub parts, we will provide the solution only to the first
three sub parts as per our Q&A guidelines. Please repost the remaining sub parts separately.
Step 2
3. (a) The Lineweaver Burk Plot for C₂H5OH in presence and absence of inhibitor is as follows:
[$]
Vo (no hinhibitor) Vo (with inhibitor) 1/[5]
1/Vo (no inhibitor)
1/Vo (with inhibitor)
0.008
0.002
0.001
0.00067
0.00011
0.000056
0.000033
0.000017
--62000
0.008
0.002
0.001
0.00067
2.1
1.8
1.5
1.35
4
0.00011
0.000056
0.000033
0.000017
-1/Km
-1/Km-1600
=> Km = 1/1600
1.7
14
1.3
0.95
-42000
The Lineweaver Burk Plot for NAD* in presence and absence of inhibitor is as follows:
[s]
Vo (no inhibitor) Vo (with inhibitor)
1/[5]
1/Vo (no inhibitor)
-1/Km
↓
-62000
-1600-1400-1200-1000 -800 60400-2000 200 400 600 800 1000 1200 1400 1600 1800
1/[5]
2.1
1.8
1.5
1.35
1.7
1
0.69
0.52
-22000
1.7
2
1.3
0.95
2.5
-42000
0.85
0.71
0.63
0.47
1.5
-22000
0.5
25
2
35
As
2
1
0
-2000
Step 3
(b) From the Lineweaver Burk plot, we can find the Km. For C₂H5OH, the graph will be:
[S]
Vo (no hinhibitor) Vo (with inhibitor)
1/[5]
1/Vo (no inhibitor)
1.7
0.69
0.52
1
0.85
0.71
2.5
-1/km
0.63
0.47
0.5
125
500
1000
1492.537313
2.5
1/[5]
1
2
5
Os
-1/Vo (no inhibitor)
-1/Vo (with inhibitor)
9090.909091
17857.14286
30303.0303
58823.52941
1/Vo (no inhibitor)
1/Vo (with inhibitor)
=> Km = 0.000625 M
Therefore, Km for C₂H5OH will be 0.000625 M.
From the Lineweaver Burk plot, we can find the Km. For NAD+, the graph will be:
[s]
Vo (no inhibitor)
Vo (with inhibitor)
1/[S]
1/Vo (no inhibitor)
0
-2000
18000
0
-1600-1400-1200-1000-800 60400-200 0 200 400 600 800 1000 1200 1400 1600 1800
4
1/[S]
1492.537313
125
500
1000
From graph, we can see that the line intersects X-axis at a point. In case of C₂H5OH when no inhibitor is
used,
1/Vmax
WAS THIS HELPFUL?
38000
0.476190476
0.555555556
0.666666667
0.740740741
9090.909091
17857.14286
30303.0303
58823.52941
→1/Vo (no inhibitor)
➡1/Vo (with inhibitor)
+1/Vo (no inhibitor)
1/[5]
1/Vo (with inhibitor)
1/Vmax
0.588235294
0.714285714
0.769230769
1.052631579
1/Vmax
58000
18000
0.476190476
0.555555556
0.666666667
0.740740741
38000
1/Vo (with inhibitor)
0.588235294
1.449275362
1.923076923
0.588235294
0.714285714
0.769230769
1.052631579
58000
1.176470588
1.408450704
1.587301587
2.127659574
78000
1
1/Vo (with inhibitor)
0.588235294
1.449275362
1.923076923
1/Vo (with inhibitor)
1
1.176470588
1.408450704
78000
1.587301587
2.127659574
From graph, we can see that the line intersects X-axis at a point. In case of NAD+ when no inhibitor is
used,
-1/Km =-62000
=> Km = 1/62000
=> Km = 0.00001613 M
Therefore, Km for NAD+ will be 0.00001613 M.
Step 4
(c) To find the type of inhibition, we have to observe the Km. We know the Km for C₂H5OH is 0.000625 M.
The Km for C₂H5OH in presence of inhibitor will be:
1/Km = -400
=>Km=1/400
=>Km = 0.0025 M.
Since, the Km of C₂H5OH in presence of inhibitor is more than the Km of C₂H5OH in absence of inhibitor,
the inhibition is competitive inhibitor.
In competitive inhibitor, the inhibitor is binding to the substrate binding site. As a result, maximum
velocity decreases. Thus, higher concentration of substrate is required to attain maximum velocity in
presence of inhibitor. Hence, the km increases.](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fd2705ec0-2034-4d28-a385-06c28bb62d2f%2F017e1983-11c6-46c7-ab0d-29d67e34de55%2F4l65ps2_processed.png&w=3840&q=75)
Transcribed Image Text:Step 1
Since you have posted a question with multiple sub parts, we will provide the solution only to the first
three sub parts as per our Q&A guidelines. Please repost the remaining sub parts separately.
Step 2
3. (a) The Lineweaver Burk Plot for C₂H5OH in presence and absence of inhibitor is as follows:
[$]
Vo (no hinhibitor) Vo (with inhibitor) 1/[5]
1/Vo (no inhibitor)
1/Vo (with inhibitor)
0.008
0.002
0.001
0.00067
0.00011
0.000056
0.000033
0.000017
--62000
0.008
0.002
0.001
0.00067
2.1
1.8
1.5
1.35
4
0.00011
0.000056
0.000033
0.000017
-1/Km
-1/Km-1600
=> Km = 1/1600
1.7
14
1.3
0.95
-42000
The Lineweaver Burk Plot for NAD* in presence and absence of inhibitor is as follows:
[s]
Vo (no inhibitor) Vo (with inhibitor)
1/[5]
1/Vo (no inhibitor)
-1/Km
↓
-62000
-1600-1400-1200-1000 -800 60400-2000 200 400 600 800 1000 1200 1400 1600 1800
1/[5]
2.1
1.8
1.5
1.35
1.7
1
0.69
0.52
-22000
1.7
2
1.3
0.95
2.5
-42000
0.85
0.71
0.63
0.47
1.5
-22000
0.5
25
2
35
As
2
1
0
-2000
Step 3
(b) From the Lineweaver Burk plot, we can find the Km. For C₂H5OH, the graph will be:
[S]
Vo (no hinhibitor) Vo (with inhibitor)
1/[5]
1/Vo (no inhibitor)
1.7
0.69
0.52
1
0.85
0.71
2.5
-1/km
0.63
0.47
0.5
125
500
1000
1492.537313
2.5
1/[5]
1
2
5
Os
-1/Vo (no inhibitor)
-1/Vo (with inhibitor)
9090.909091
17857.14286
30303.0303
58823.52941
1/Vo (no inhibitor)
1/Vo (with inhibitor)
=> Km = 0.000625 M
Therefore, Km for C₂H5OH will be 0.000625 M.
From the Lineweaver Burk plot, we can find the Km. For NAD+, the graph will be:
[s]
Vo (no inhibitor)
Vo (with inhibitor)
1/[S]
1/Vo (no inhibitor)
0
-2000
18000
0
-1600-1400-1200-1000-800 60400-200 0 200 400 600 800 1000 1200 1400 1600 1800
4
1/[S]
1492.537313
125
500
1000
From graph, we can see that the line intersects X-axis at a point. In case of C₂H5OH when no inhibitor is
used,
1/Vmax
WAS THIS HELPFUL?
38000
0.476190476
0.555555556
0.666666667
0.740740741
9090.909091
17857.14286
30303.0303
58823.52941
→1/Vo (no inhibitor)
➡1/Vo (with inhibitor)
+1/Vo (no inhibitor)
1/[5]
1/Vo (with inhibitor)
1/Vmax
0.588235294
0.714285714
0.769230769
1.052631579
1/Vmax
58000
18000
0.476190476
0.555555556
0.666666667
0.740740741
38000
1/Vo (with inhibitor)
0.588235294
1.449275362
1.923076923
0.588235294
0.714285714
0.769230769
1.052631579
58000
1.176470588
1.408450704
1.587301587
2.127659574
78000
1
1/Vo (with inhibitor)
0.588235294
1.449275362
1.923076923
1/Vo (with inhibitor)
1
1.176470588
1.408450704
78000
1.587301587
2.127659574
From graph, we can see that the line intersects X-axis at a point. In case of NAD+ when no inhibitor is
used,
-1/Km =-62000
=> Km = 1/62000
=> Km = 0.00001613 M
Therefore, Km for NAD+ will be 0.00001613 M.
Step 4
(c) To find the type of inhibition, we have to observe the Km. We know the Km for C₂H5OH is 0.000625 M.
The Km for C₂H5OH in presence of inhibitor will be:
1/Km = -400
=>Km=1/400
=>Km = 0.0025 M.
Since, the Km of C₂H5OH in presence of inhibitor is more than the Km of C₂H5OH in absence of inhibitor,
the inhibition is competitive inhibitor.
In competitive inhibitor, the inhibitor is binding to the substrate binding site. As a result, maximum
velocity decreases. Thus, higher concentration of substrate is required to attain maximum velocity in
presence of inhibitor. Hence, the km increases.
Expert Solution
This question has been solved!
Explore an expertly crafted, step-by-step solution for a thorough understanding of key concepts.
This is a popular solution!
Trending now
This is a popular solution!
Step by step
Solved in 3 steps with 1 images
Recommended textbooks for you
Biochemistry
Biochemistry
ISBN:
9781305577206
Author:
Reginald H. Garrett, Charles M. Grisham
Publisher:
Cengage Learning
Biology: The Dynamic Science (MindTap Course List)
Biology
ISBN:
9781305389892
Author:
Peter J. Russell, Paul E. Hertz, Beverly McMillan
Publisher:
Cengage Learning
Biochemistry
Biochemistry
ISBN:
9781305577206
Author:
Reginald H. Garrett, Charles M. Grisham
Publisher:
Cengage Learning
Biology: The Dynamic Science (MindTap Course List)
Biology
ISBN:
9781305389892
Author:
Peter J. Russell, Paul E. Hertz, Beverly McMillan
Publisher:
Cengage Learning