4. Based on the data in Figure 1, estimate the maximal velocity (Vm) and the Mic

Question

4. Based on the data in Figure 1, estimate the maximal velocity (Vm) and the

Michaelis-Menten constant (Km) for alkaline phosphatase.

Figure 1. Substrate concentration-velocity curve for alkaline phosphatase based on means and error bars from five replicate groups. Enzyme concentration in all reactions was 1.5 mg/100 ml.

Vm =

Km =

5. You conduct an experiment to determine the action of lipase in the presence of milk. Milk contains protein, sugar and fat.

You prepare 3 test tubes as shown below adding equal amounts of phenolphthalein as a pH indicator to each tube. Phenolphthalein is pink in basic solutions and clear in acidic solutions. You confirm the addition of Na2CO3 to each tube makes the beginning solutions basic as indicated by their pink color.

Test

Tube

Milk

(mL)

Na2CO3

(mL)

3% bile salts

(mL)

5% lipase

(mL)- boiled

5% lipase

(mL)- unboiled

1

5

7

none

None

1

2

5

7

1

1

None

3

5

7

1

none

1

Results:

Test

Tube

Milk

(mL)

Na2CO3

(mL)

Additional reactants

Time to change from pink to white (sec)

1

5

7

Lipase only

400

2

5

7

Boiled lipase and bile salts

Never

3

5

7

Lipase and

bile salts

200

a. What products would you expect to find from the digestion of milk (Hint: Think about the monomers or components of each of the substances listed)?

b. What could account for the color (pH) change in the test tubes?

c. Which part of the experiment suggests that lipase acts as an enzyme?

d. From the results shown do bile salts contain an enzyme that affects milk? Explain your reasoning.

Test

Tube

Milk

(mL)

Na2CO3

(mL)

3% bile salts

(mL)

5% lipase

(mL)- boiled

5% lipase

(mL)- unboiled

1

5

7

none

None

1

2

5

7

1

1

None

3

5

7

1

none

1

Explanation / Answer

5.

a) Lipase is an enzyme that breakdown lipids into fatty acids and glycerol. So, the digestion of milk result in the formation of fatty acids and glycerol.

b). The colour change from pink to colourless (appears white as the sample is milk in this case), indicates the increased acidity of the sample, which is due to the increased levels of fatty acids and glycerol.

c). The second and third test tubes indicates that the lipase is acting as an enzyme. Because in the presence of boiled lipase (denatured), bile salts fail to digest milk, where as in the presence of lipase, they did. This indicates that lipase act as an enzyme.

d). From the results given we can say that bile salts do not contain an enzyme that affects milk, they need lipase for milk digestion. Because boiled bile salts did not lose their activity, whereas lipase lost its activity upon heating (it is an enzyme).

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