BIOL analytical assignment

Define specific activity and indicate units.

The activity of an enzyme per milligram of total protein (expressed in ìmol/time/mg of all protein).

Addition of which metal ion or combination of ions restores alkaline phosphatase activity closest to that of native enzyme?

The addition of Mg, Zn gets the specific activity closest to that of the native enzyme

What reaction does alkaline phosphatase catalyze? Identify the substrate (type of molecule), any other reactants and the product(s).

The enzyme alkaline phosphatase has the ability to catalyze different ranges of phosphate esters, specifically it can be used in numerous reactions such as the hydrolysis of monoesters of phosphoric acid. The enzyme can also be used to dephosphorylate certain compounds so that these molecules can freely diffuse into the cell.

Which ion is least likely to act as a co-factor of native alkaline phosphatase? Explain your reasoning.

The ion least likely to act as a co-factor is Ca since it has the lowest specific activity out of all of them.

What effect did removal of all metal ions have compared to native alkaline phosphatase activity?

The native ALP had a much higher specific activity than all the other metals except for the addition of Cobalt.

Based on your answer to Question 5, predict the pH range at which this enzyme functions in its natural environment.

The pH range the enzyme functions in its natural environment is probably 8.5-9.5

a) What was the pH of the reaction mixture used for the temperature experiment? b) Why did the researchers choose to use this pH?

a) pH: 9.5 b) the researchers chose to use this pH because it restores pH balance by reducing acidity levels

Some of the metal ions tested have a positive effect on enzyme activity. What are these types of positive regulators called?

co factors

In the metal ion experiment, which condition is the control and which condition could be considered a negative control?

control: native AP negative control: metal free AP w/o any metal added

Define metalloprotein.

large group of proteins containing one or more atoms of metal bound to specific sites in the polypeptide chain

What is the maximum specific activity measured for this enzyme and at what pH does it occur?

maximum specific activity: 0.325 umol/min/mg pH: 9.5

Over what pH range does the enzyme have 50 - 100% of its maximum specific activity?

pH range: 8.5-10

Explain why the researchers added EDTA to the solution. Use additional references to provide specific information about which metals interact with EDTA.

the researchers added EDTA to the solutions to help bind metal ions in aqueous solutions. metal examples: calcium, magnesium, lead, and iron

Predict the temperature range at which the bacteria live.

40-80 degrees C

Express the activity of the metal-free enzyme as a percentage of the activity of the native enzyme.

5.4%

Over what range of temperatures does the enzyme have 50 - 100% of its maximum specific activity?

60-80 degrees C

At what temperature does maximum specific activity occur?

80 degrees C

What organisms make alkaline phosphatase enzymes? Do some additional research to answer this question.

Bacteria makes up alkaline phosphatase enzymes (can be found in E. coli)

Using your knowledge of pH effects on protein function, what is likely to happen to this enzyme at pH 4.5?

If the enzyme was put into a pH of 4.5, the specific activity would dramatically decrease since the enzyme will be denatured.