Scribd
Upload
45 views

Biochemistry PRC 2 Data Analysis Enzyme

The document discusses factors affecting enzyme activity and kinetics. It provides data and questions about enzyme kinetics experiments involving determination of Km, Vmax, types of inhibition, and effects of various factors on reaction rate.

Uploaded by

SARAH SAFIAH TAJUL ARIFFIN
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
45 views

Biochemistry PRC 2 Data Analysis Enzyme

The document discusses factors affecting enzyme activity and kinetics. It provides data and questions about enzyme kinetics experiments involving determination of Km, Vmax, types of inhibition, and effects of various factors on reaction rate.

Uploaded by

SARAH SAFIAH TAJUL ARIFFIN
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 4

PRACTICAL 2:

DATA ANALYSIS IN ENZYMOLOGY

Specific Learning Outcomes


After this practical the students should be able to understand and

Knowledge:
1. discuss the factors affecting enzyme activity
2. discuss the effect of competitive and non-competitive inhibitor toward enzyme
kinetics (Km and Vmax)
3. Discuss the differences between Michaelis Menten enzyme and allosteric enzyme.

Skills:
1. know how to draw velocity vs substrate graph and Lineweaver Burk graph.
2. know how to determine Km and Vmax from graph velocity vs substrate graph and
Lineweaver Burk Graph.

Attitude:
1. develop communication skill within team members
2. inculcate teamwork
Problem Solving

1.The absorbance shown in the table were obtained after p-nitrophenolphosphate


was mixed with buffer and serum sample. The colorless mixture turns into yellow after
incubation. The absorbance was measured using spectrophotometer at 405 nm.

ALP
p-nitrophenolphosphate + H2O + Mg2+ ----→ p-nitrophenol + phosphate

Absorbance at minute)
0 2 3 4 5 6 7
0.144 0.18 0.22 0.25 0.28 0.31 0.34

Answer the following question:

a. Why does the colorless mixture turn into yellow after several minutes?

b. What is the assumption made in measuring ALP activity.

c. Calculate Delta absorbance for each time point.

d. Using formula (V=2742 x Delta absorbance/ min at 405 nm), find the velocity
of the enzyme and plot graph velocity versus time.

2. The data shown below were obtained for an enzyme-catalyzed reaction. Using
Michaelis-Menten graph determine the value for Km and Vmax

[S](mM) V (mmol ml-¹ min-¹)

0.1 3.33

0.2 5.00

0.5 7.14

0.8 8.00

1.0 8.33

2.0 9.09
3. You are provided data compiled from kinetic analysis of an esterase enzyme. The
Enzyme catalyzes the hydrolysis of an ester called methylbenzoate. Use the
Michaelis-Menten and Lineweaver-Burk graphs to determine Km and Vmax. Include
the proper units for each constant. Compare your results for each graphical
method. The data were obtained by measuring the rate of disappearance of
substrate.

Methyl benzoate V0
(nmol/L) (nmol/min)
3.7 10
13.0 30
39.0 63
79.0 87
230.0 116

4. The data shown below were obtained for an enzyme-catalyzed reaction in the
presence and absence of inhibitor X. State and explain the type of inhibition that
has occurred. (You can use Michaelis-Menten or Lineweaver-Burk graph to answer
this question)

V (mmol ml-¹ min-¹)

[S] (mM) without X with X

0.2 5.0 3.0

0.4 7.5 5.0

0.8 10.0 7.5

1.0 10.7 8.3

2.0 12.5 10.7

4.0 13.6 12.5


5. Which of the following factors will influence the reaction rate for a typical
enzyme? Will the change increase, decrease, or have no effect on the reaction
rate?

(a) Increase substrate concentration.


(b) Increase the temperature from 25ºC.to 37ºC.
(c) Add a competitive inhibitor
(d) Change the pH from 7 to 1.
(e) Increase the temperature from 37ºC to 150ºC.
(f) Increase the enzyme concentration.

6. The velocity of an enzyme catalyzed reaction was plotted against the substrate
concentration and the data obtained is shown below. Plot a graph of the Velocity
(V) against the substrate (S) concentration and explain the kinetic behavior of the
enzyme. What is the substrate concentration at half maximum velocity?

Substrate (Reaction velocity)


umol/min
mM

1.0 0.08

2.0 0.20

3.0 0.60

4.0 1.20

5.0 1.64

6.0 1.80

7.0 1.90

8.0 1.98

9.0 2.00

10.0 2.10

You might also like