TURN OVER 1

This paper consists

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.

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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
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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

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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















END