1. The following data were obtained from an enzyme kinetics experiment.  Graph the data using a Lineweaver-Burk plot and determine, by inspection of the graph, the values for K_m and V_max.

[S] (µM)               V (nmol/min)

_______               ___________

0.20                                              1.43

0.26                                              1.67

0.33                                              2.08

1.00                                              3.33

 

2. Calculate the specificity constant for an enzyme if its k_cat = 1.4 x 10⁴ s^-1 K_m = 90 µM.  Is this enzyme very efficient?

 

3. Use the Michaelis-Menton Equation to calculate the missing values of [S] given below if V_max = 5 mmol/min.  Plot [S] versus V (NOT the reciprocals!).  Draw line parallel to the x-axis at V_max and extend your plotted line to show its approach to V_max.

[S] (mM)              V (mmol/min)

_______               ___________

10                              1.2

?                           1.7

?                           2.1

?                           2.2

?                           2.5

 

4.  The effect of an inhibitor on an enzyme was tested and the experiment gave the results below.  Plot the data and determine, by inspection of the graph, what type of inhibition is involved.

[S] µM      V (µmol/min)  V (µmol/min)  V (µmol/min)

                 with 0.0 nM    with 25 nM     with 50 nM

                 Inhibitor          Inhibitor          Inhibitor

______     ___________  ___________  ___________

                     0.4          0.22                0.21                 0.20

                     0.67        0.29                0.26                 0.24

                     1.00        0.32                0.30                 0.28

                     2.00        0.40                0.36                 0.32

5. You perform a kinetics experiment on the enzyme phosphatidylinositol synthase (PI synthase) using as substrate radiolabeled inositol in a tracer amount mixed with unlabeled inositol

                                                               PI Synthase

      CDP-DAG + Inositol —————————–à  Phosphatidylinositol

Using a scintillation counter, you have determined that the inositol substrate has a specific radioactivity of 100 dpm (disintegrations per minute) / 1 nmol of inositol.

You collect the following data that represent the amount of radiolabeled phosphatidylinositol formed (data in dpm) after a 10 min reaction using 5 µM enzyme.

[S]  nmol/100 µl Reaction Volume                dpm Recovered in Product Formed

____________________________               ____________________________

            250                                                           1,000

500                                                                                                                        16,000

750                                                                                                                        17,500

1,000                                                        21,700

 

1. Determine nmol product formed per min for each substrate concentration used.

2. Prepare a Lineweaver-Burk plot of the data.

3. Determine K_m and V_max for PI synthase.

6. (a)  Define Molar (M)

(b)  Define mol (or mole)