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How do you know that the change in the absorbance at 340 nm that you measure in this experiment is due to the activity of alcohol dehydrogenase and not to the activity of some other enzyme that uses NAD+ as a coenzyme?

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Collen Von
Collen VonLv2
28 Sep 2019

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Related questions

6. It is your desire to purify the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the common sparrow. The enzyme has a MW of ~35kDa and a theoretical pI of ~9.00. In addition the enzyme carries out the following reaction: Glyceraldehyde-3-phosphate + NAD+ + Pi ↔1,3 bisphosphoglycerate + NADH

a. Based on this information would a Q-sepharose column be a good ion-exchanger for this protein (1 point)? Why or why not (1 point)?

b. Would you expect this protein to bind to a HiTrapTM Blue HP column? Why or why not (1 points)?

c. If you wanted to do an enzymatic activity assay on this protein using the absorbance properties of NAD+/NADH at 340 nm, what substrate would you put in the reaction mixture: Glyceraldehyde-3-phosphate or 1,3-bisphosphoglycerate (2 points)? Why (2 points)? (NOTE: You want absorbance at 340 nm to increase throughout the assay)

d. The last step of your purification protocol involves running your protein sample over a size exclusion column. At first you choose a column with a molecular weight cutoff of 80 kDa. Due to the size of your protein you expect it to come off somewhere in the middle of the run and foolishly decide to start collecting fractions half-way through the run. When you come back you see your data and realize the protein came off in the “void” volume (very early in the run) suggesting that your protein is much larger than 80 kDa. What is a possible explanation for these data? (1 point)

Although both pyruvate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase use the same oxidant as their electron acceptor, the two enzymes do not draw from the same cellular oxidant pool. Why? Choose ONE answer.

Question options:

NAD+ is only used once before being degraded, so it is not regenerated for subsequent rounds

The NAD+ pool in the mitochondria and cytosol are separated by the mitochondrial membrane

The enzymes are found in different types of cells Both enzymes use FAD as their electron acceptors.

The pyruvate dehydrogenase reaction uses FAD as its sole electron acceptor whereas glyceraldehyde 3-phosphate dehydrogenase uses NAD+

NAD+ remains tightly bound to each enzyme, so the NAD pools are not shared.

1. Choose the TWO correct statements describing the citrate cycle.

ATP is an indirect product of the citric acid cycle

Acetyl CoA can increase the pool of citric acid cycle intermediates

Fatty acids can increase the pool of citric acid cycle intermediates

ADP inhibits α-ketoglutarate dehydrogenase

The pool of mitochondrial oxaloacetate can be increased by pyruvate

All enzymes of the citric acid cycle are found soluble and free in the mitochondrial matrix

2.Which molecule in the net reaction of the citrate cycle contributes to the inhibition of pyruvate dehydrogenase?

NADH

H+

H2O

FAD

3. How would a high NADH to NAD+ ratio be expected to affect the pyruvate dehydrogenase reaction?

The E1 subunit is phosphorylated by pyruvate dehydrogenase kinase, and the catalytic activity of pyruvate dehydrogenase decreases.

The last step of the pyruvate dehydrogenase reaction is blocked, resulting in a decrease in activity.

The pyruvate dehydrogenase phosphatase-1 enzyme would increase, resulting in pyruvate dehydrogenase activation at an accelerated rate.

The pyruvate dehydrogenase kinase enzyme activity would increase, resulting in an increase in pyruvate dehydrogenase activity.

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