Malate dehydrogenase is an enzyme that converts malate to oxaloacetate in the last stage of the TCA Cycle (citric acid cycle/Krebs cydle), represented by the following equation: NAD+ Malate Oxaloacetate NADH A group of students carried out an experiment to determine the optimum temperature for the activity of a commercially produced malate dehydrogenase (from yeast) at several different temperatures. A series of test tubes was set up containing 2.0 cm of phosphate buffer with a pH of 7.5, 0.1 cm? of NADH, 0.1 cm3 of malate dehydrogenase and 0.7 cm3 of water. The tubes were incubated in water baths at the various temperatures for 5 minutes. At certain time intervals each tube was placed in a colorimeter, set at an absorbance value of 1.0. The reaction was then started by adding 0.1 cm? of oxaloacetic acid to the tube. Enzyme activity was measured by following the decrease in absorbance for 120 seconds. The experiment was repeated to give duplicate results. Enzyme activity was then calculated as pmoles per mg of (enzyme) protein. The results are shown in Table 1 below: Table 1: Results to show the activity of malate dehydrogenase at various temperatures. Enzyme activity (umoles per mg protein) Temp. (°C) Average (to two decimal points 0.20 12.90 Test 2 (Duplicate test) Test 1 0.20 0.20 12.7 0.00 10.0 13.0 20.0 30.0 40.0 52.0 91.3 82.7 50.2 90.0 79.3 51.10 90.70 81.00 40.80 16.60 50.0 41.5 40.0 60.0 70.0 17.2 16.0 9.90 10.0 9.95 a) On the graph paper provided, plot the data in a suitable form. b) Briefly explain the effect of temperature on malate dehydrogenase activity. What is the optimum temperature for the activity of malate dehydrogenase (calculated from your graph)? c) d) The value you have calculated in (c) above may not be the true optimum temperature for the enzyme. Describe how you could modify the experiment to get a more accurate optimum value. Why were the tubes incubated for 5 minutes prior to the addition of oxaloacetic acid to start the reaction? e)
Malate dehydrogenase is an enzyme that converts malate to oxaloacetate in the last stage of the TCA Cycle (citric acid cycle/Krebs cydle), represented by the following equation: NAD+ Malate Oxaloacetate NADH A group of students carried out an experiment to determine the optimum temperature for the activity of a commercially produced malate dehydrogenase (from yeast) at several different temperatures. A series of test tubes was set up containing 2.0 cm of phosphate buffer with a pH of 7.5, 0.1 cm? of NADH, 0.1 cm3 of malate dehydrogenase and 0.7 cm3 of water. The tubes were incubated in water baths at the various temperatures for 5 minutes. At certain time intervals each tube was placed in a colorimeter, set at an absorbance value of 1.0. The reaction was then started by adding 0.1 cm? of oxaloacetic acid to the tube. Enzyme activity was measured by following the decrease in absorbance for 120 seconds. The experiment was repeated to give duplicate results. Enzyme activity was then calculated as pmoles per mg of (enzyme) protein. The results are shown in Table 1 below: Table 1: Results to show the activity of malate dehydrogenase at various temperatures. Enzyme activity (umoles per mg protein) Temp. (°C) Average (to two decimal points 0.20 12.90 Test 2 (Duplicate test) Test 1 0.20 0.20 12.7 0.00 10.0 13.0 20.0 30.0 40.0 52.0 91.3 82.7 50.2 90.0 79.3 51.10 90.70 81.00 40.80 16.60 50.0 41.5 40.0 60.0 70.0 17.2 16.0 9.90 10.0 9.95 a) On the graph paper provided, plot the data in a suitable form. b) Briefly explain the effect of temperature on malate dehydrogenase activity. What is the optimum temperature for the activity of malate dehydrogenase (calculated from your graph)? c) d) The value you have calculated in (c) above may not be the true optimum temperature for the enzyme. Describe how you could modify the experiment to get a more accurate optimum value. Why were the tubes incubated for 5 minutes prior to the addition of oxaloacetic acid to start the reaction? e)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
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answer the last 2 questions d and c
Transcribed Image Text:Malate dehydrogenase is an enzyme that converts malate to oxaloacetate in
the last stage of the TCA Cycle (citric acid cycle/Krebs cydle), represented by
the following equation:
NAD+
Malate
Oxaloacetate
NADH
A group of students carried out an experiment to determine the optimum
temperature for the activity of a commercially produced malate dehydrogenase
(from yeast) at several different temperatures. A series of test tubes was set
up containing 2.0 cm of phosphate buffer with a pH of 7.5, 0.1 cm? of NADH,
0.1 cm3 of malate dehydrogenase and 0.7 cm3 of water. The tubes were
incubated in water baths at the various temperatures for 5 minutes. At certain
time intervals each tube was placed in a colorimeter, set at an absorbance
value of 1.0. The reaction was then started by adding 0.1 cm? of oxaloacetic
acid to the tube. Enzyme activity was measured by following the decrease in
absorbance for 120 seconds. The experiment was repeated to give duplicate
results. Enzyme activity was then calculated as pmoles per mg of (enzyme)
protein. The results are shown in Table 1 below:
Table 1: Results to show the activity of malate dehydrogenase at various
temperatures.
Enzyme activity (umoles per mg protein)
Temp.
(°C)
Average (to
two decimal
points
0.20
12.90
Test 2 (Duplicate
test)
Test 1
0.20
0.20
12.7
0.00
10.0
13.0
20.0
30.0
40.0
52.0
91.3
82.7
50.2
90.0
79.3
51.10
90.70
81.00
40.80
16.60
50.0
41.5
40.0
60.0
70.0
17.2
16.0
9.90
10.0
9.95
a)
On the graph paper provided, plot the data in a suitable form.
b)
Briefly explain the effect of temperature on malate dehydrogenase
activity.
What is the optimum temperature for the activity of malate
dehydrogenase (calculated from your graph)?
c)
d)
The value you have calculated in (c) above may not be the true
optimum temperature for the enzyme. Describe how you could modify
the experiment to get a more accurate optimum value.
Why were the tubes incubated for 5 minutes prior to the addition of
oxaloacetic acid to start the reaction?
e)
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