Download PTT 103 BIOCHEMISTRY SEMESTER I, 2013/2014 ENZYMES

PTT 103 BIOCHEMISTRY
SEMESTER I, 2013/2014
ENZYMES, NUCLEIC ACIDS AND GENETIC INFORMATION
Submission date: 16/12/13
1. Consider the following data for an enzyme-catalyzed hydrolysis reaction by the
inhibitor I:
[Substrate] (M)
v (µmol/min)
v1 (µmol/min)
6 x 10-6
20.8
4.2
1 x 10-5
29
5.8
2 x 10-5
45
9
6 x10-5
67.6
13.6
1.8 x 10-4
87
16.2
a) Using a Michaelis-Menten plot, determine Km for the uninhibited reaction and the
inhibited reaction.
b) Generate a Lineweaver-Burk plot for the data.
c) Explain the significance of the
i)
Horizontal intercept: -1/Km
ii)
Vertical intercept: 1/Vmax
iii)
Slope: Km/Vmax
d) What type of inhibition is being measured.
- Type of inhibition is noncompetitive.
2. When DNA is heated, it denatures, that is the strands separate. Determine which of the
following molecules will denature first as the temperature is raised. Why?
5’- GCATTTCGGCGCGTTA-3’
3’- CGTAAAGCCGCGCAAT-5’
5’- ATTGCGCTTATATGCT-3’
3’- TAACGCGAATATACGA-5’
-
The G-C base pair with its 3 H bonds is more stable than the A-T base pair.
The more CG bp there are, the more stable the DNA molecule
Structure B with the least number of G-C base pair will denature first.
3. Human insulin gene can be isolated from genomic DNA library and screened for
positive clones using plaque hybridization method.
i) Demonstrate the steps involved in the construction of genomic DNA library.
ii) Demonstrate plaque hybridization method.
4. List and describe the steps in prokaryotic DNA replication.
5. Human insulin can be produced in E. coli using recombinant DNA technology.
Demonstrate the recombinant DNA technique.