of 4 pages, each of which is identified by the code number BIOC130301 Battery operated, non-programmable calculators allowed. An approved basic translation dictionary is allowed. 2 sheets of graph paper required.
FACULTY OF BIOLOGICAL SCIENCES UNDERGRADUATE SCHOOL
BIOCHEMISTRY AND MICROBIOLOGY
UNIVERSITY OF LEEDS
May 2010
BIOC 1303: Introductory Biochemistry: Problem Solving and Data Handling
Time allowed: 2 hours
This examination paper comprises two sections, each section carries equal weighting.
You should attempt ALL questions in both SECTION A and SECTION B.
Answer each Section in a separate answer book.
SECTION A
1. Describe how you would make 500 ml of a solution containing 50 mM sodium citrate, 1 mM dithiothreitol and 0.02% (w/v) Triton X-100 (a detergent).
Triton X-100 is available as a 10% (w/v) stock). The molecular mass of sodium citrate is 236.1 Da. The molecular mass of dithiothreitol is 154.2 Da.
2. Efficient ligation of a 500 bp DNA fragment with a linearised plasmid vector requires a 10:1 molar ratio of fragment to vector molecules. The DNA fragment is available as a stock solution that has an absorbance of 0.8 at 260 nm in a 1 cm path length cuvette. Calculate the volume of the DNA fragment solution that should be included in a ligation reaction mixture that contains 20 fmol of vector.
The average molecular mass of a nucleotide base-pair is 660 Da. A 50 ng/l solution of double stranded DNA has an absorbance of 1 at 260 nm in a cuvette with a path length of 1cm. 1 fmol =10 -15 mol.
BIOC 130301
TURN OVER 2 3. The structure of an amino-acid is shown below. The pKs of the and R-carboxyl groups and the -amino group are 2.1, 3.9 and 9.8, respectively.
(i) Name the amino-acid.
(ii) Draw the predominant ionic species at pH 1.0 and at pH 11.0.
(iii) What is the pI of this amino-acid?
(iv) Draw the predominant ionic species at the pI.
4. The folded conformation of the bacterial protein Im9 is 16.1 kJ mol -1 more stable than the unfolded state at 10C. Calculate the percentage of molecules in the folded state at this temperature.
R =8.31 J K -1 mol -1 . 0C =273.15 K.
N H 2 CH 2 H COOH COOH BIOC 130301
TURN OVER 3 SECTION B
A researcher aims to carry out the reduction of a molecule used in the synthesis of a drug precursor using a non-specific dehydrogenase (DH).
Due to the properties of the drug precursor, reaction conditions of 70C and pH 3.0 must be used. The enzyme will need to be an efficient catalyst under these conditions.The researcher therefore decides to isolate and purify the dehydrogenase from a thermophilic bacterium. The molecular mass of this enzyme is 36,724 Da.
1. Sketch a graph showing the relationship between enzyme activity and temperature that you expect for this enzyme.
2. Indicate on your sketch how this relationship might differ from that for the same enzyme isolated from rat skeletal muscle.
3. Based on your sketch, explain why the company chose to extract the lactate dehydrogenase from a thermophilic bacterium.
In experiments to investigate the suitability of the enzyme the researcher measured its kinetic properties. Table 1 shows the results of an experiment to determine the effect of pH on maximum velocity.
Table 1
[S] mM v o pH 3.0 (mol min -1 DH mg -1 ) v o pH 7.0 (mol min -1 DH mg -1 ) 0.10 0.10 0.39 0.25 0.23 0.91 0.75 0.58 2.26 1.50 0.91 3.60 7.50 1.71 6.83 10.00 1.81 7.24
(i) On the graph paper provided, draw a suitable graph to determine the maximum velocities of the enzyme-catalysed reactions at pH 3.0 and 7.0. Show how you derived the V max values. Attach your graph to this answer book.
(ii) Calculate k cat (in units of s -1 ) for this enzyme at pH 7.0
(iii) Calculate the percentage of enzyme activity lost upon reducing the pH. Explain whether you think the enzyme will be suitable for use at pH 3.0.
4. The amino acid sequence of the purified rat enzyme was determined and the order of residues from 101 to 108 is given below.
Glu 101 MetGlnTrpAsnLysGlnLeu 108
Using the attached copy of the genetic code write down a possible double-stranded DNA sequence for this amino-acid sequence. Show the polarities of the strands and indicate which are coding, non-coding, sense and template.
BIOC 130301
TURN OVER 4 5. A comparison of the genes for similar dehydrogenase enzymes from the thermophilic bacterium and rat muscle showed that the codons for residue 104 differed in their pyrimidine base.
(i) Show how the codons differ for residue 104 in the DNA of the two organisms.
(ii) What difference will this make to the amino acid sequences?
(iii) Compare the characteristics of these two amino acids and speculate on the effect of this difference on the tertiary structure and hence the thermal stabilities of the two enzymes.
2nd Base
U C A G
1st Base
U
Phe Phe Leu Leu
Ser Ser Ser Ser Tyr Tyr Stop Stop Cys Cys Stop Trp U C A G 3rd Base
C Leu Leu Leu Leu Pro Pro Pro Pro His His Gln Gln Arg Arg Arg Arg U C A G
A Ile Ile Ile Met Thr Thr Thr Thr Asn Asn Lys Lys Ser Ser Arg Arg U C A G
G Val Val Val Val Ala Ala Ala Ala Asp Asp Glu Glu Gly Gly Gly Gly U C A G