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Final 23 June Autumn 2020

Functional Proteins And Genes (Western Sydney University)

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FINAL EXAM – AUTUMN SESSION 2020

School of Science

Complete your details in this section at the start of the exam.

STUDENT SURNAME:

STUDENT FIRST NAME:

STUDENT ID:

EXAM INSTRUCTIONS
Read all the information below and follow the instructions carefully.

UNIT NAME: Functional Proteins & Genes

UNIT NUMBER: 300936

NUMBER OF QUESTIONS: 10 Multiple choice and 13 short answer questions

VALUE OF QUESTIONS: Each of multiple choice question is worth 1 mark - 10 marks in

total; each short answer question is worth the marks indicated
in parentheses – 50 marks in total; therefore giving an overall
total of 60 marks for this final exam.

ANSWERING QUESTIONS: Answer multiple choice questions by circling the answer on the
exam paper or typing your choice into a Word document.
Answer short answer questions in space provided on the exam
paper or typing into a Word document.

LECTURER/UNIT Dr Ming J. Wu
COORDINATOR:

TIME ALLOWED: 2 hours TOTAL 12

PAGES:

RESOURCES ALLOWED
Only the resources listed below are allowed in this exam.
This is an open book exam. Graph paper should be used for your plots.

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10 multiple choice questions, 1 mark each, answer ALL questions

__________________________________

1. Which of the following amino acids has a net positive charge at pH 7.4?
a. Arginine
b. Cysteine
c. Aspartic acid
d. Asparagine

2. Myoglobin is a compact and mainly α helical protein. The amino acids leucine
and alanine would be predicted to be:
a. buried in the core of the protein
b. available to interact with the aqueous environment
c. involved in disulfide bond formation
d. involved in ionic interactions

3. Which of the following is true for Michaelis-Menten enzyme kinetics?

a. The lower the Km, the higher affinity of the substrate to the enzyme
b. The value forKmis independent of temperature
c. Vmax is unimportant because we measure 1/V max to find Km
d. Kmis calculated after the reaction reaches equilibrium

4. The name of an enzyme that would catalyse the formation of a

lysophospholipid from a phospholipid is?
a. Lysophosphatidyl kinase
b. Lysophosphatidyl phosphatase
c. Phospholipase C
d. Phospholipase A2

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5. What are the amino acid residues in the tripeptide below (from N-terminus to
C-terminus)?

a. Threonine, alanine and serine

b. Glycine, cysteine and alanine
c. Serine, cysteine and glycine
d. Serine, methionine and glycine

Examine the following structure (For Question 6):

1 O

H2 C O C R1
O

H2C O C R2
2 O
H2 C O P O CH2 CH2 N+ H3
O-
3 4

6. To form a diacylglycerol, hydrolysis would occur at which arrow?

a. 1
b. 2
c. 3
d. 4

7. Steady state kinetics means that the:

a. Vmax is reached when the enzyme concentration is in excess
b. Vmax is reached at the end of the assay
c. Km equals the substrate concentration
d. rate of formation of the [ES] complex is equal to its breakdown

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8. Which of the following statements is not true for the Krebs cycle?
a. It generates intermediate metabolites for the synthesis of other organic
compounds
b. It produces ATP
c. It takes place in the cytosol
d. It produces NADH

9. Cation exchange chromatography was used to separate lysine (pI 9.74) and
tyrosine (pI 5.66) in a mixture. After the mixture was applied to the cation
exchange column in citrate buffer (pH 3.75), the column was washed with the
same buffer. The elution was then carried out with Tris-HCl buffer (pH 8.8), which
amino acid(s) was (or were) eluted out of the column?
a. Lysine
b. Tyrosine
c. Lysine and tyrosine
d. Neither of the amino acids

10. When humans fast (i.e., do not eat for 24 hr), which of the following is true?
a. Insulin is released from the liver
b. Gluconeogenesis pathway is activated in the liver
c. Ketone bodies decrease in concentration
d. Glucagon is released from muscles

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13short answer questions, marks indicated in parentheses.

Answer questions in the space provided for each question or type your answers
into a Word document.
__________________________________

 Write down your PDB protein code: …2V1X……

 Write down the protein sequence number of your amino acid: ……219……….
 Using the 3-letter code what is the name of the amino acid in this position: …
Asp…
 Write down what this amino acid was mutated to: …Isoleucine (Ile/I)
…………………….

Questions 1-7 below are related to the protein assigned to you

Question 1.What is the name of your protein?(1 mark)

Name: human RECQ-like DNA helicase (RECQL)

Question 2. Describein your own words the enzymatic function of your protein.
(Up to 25 words) (1 mark)

2V1X (RECQL or RecQ1) is implicated in preserving chromosome durability. RECQL

interacts selectively and non-covalently with DNA (DNA binding). RECQL is implicated in
a variety of methods of DNA repair and maintaining genome integrity, such as mismatch
repair, nucleotide excision repair, hydrolase activity, acting on acid anhydrides and
Holliday junctions process (controls the unwinding of the DNA helix of DNA containing
Holliday junctions). It functions through catalyzing the reaction ATP + H2O → ADP + P
and thus driving the unwinding of paired DNA and translocating in the 3' to 5' direction.
RecQ1 is depending on magnesium and ATP in untwisting DNA with a 3’ to 5’ polarity.
RecQ1 helicases can, in an ATP-independent manner, boost the annealing of
complementary ssDNA particles. However, unlike the full-length protein, the truncated
protein (RECQ1T1) loses Holiday junction unwinding and DNA-annealing actions.

Question 3. Explain the structure of your protein. (Up to 100 words) (4 marks)

Primary structure: the sequence length is 591 residues. Amino acids K119, D219 and
E220 produce water-mediated connections with Mg2+, which are crucial in ATPase
function. D379 is involved in forming a single hydrogen bond between domain D2 and
ADP.

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Secondary structure: RECQL is composed of about 40% helical (24 helices; 239
residues) and 15% beta sheet (18 strands; 94 residues)
Tertiary structure: the tertiary structure has three layers.
 The top portion consisting of two RecA-like domains (D1 and D2), D1 are α-
helices, whereas D2 composed of α-helices and parallel β-sheet. These domains
are the signature motifs of helicase superfamily 2 and are believed to bear the
ATP-dependent translocation activity.
 Active side: the ADP-Mg binding site, where the DNA is absent, is located in a
deep split between the two RecA-like domains (D1 and D2) and encompassed by
extremely conserved residues. The ADP produces remarkable connections with
domain D1, but less with domain D2. The glycine-rich loop (motif 1) promotes the
phosphates.
 RecQ-Specific Zn Domain: the zinc domain (amino acids 419–480) lies below
D2 motif, and is a preserved mark of the RecQ family. It includes a 4-cysteine
zinc-binding motif, linked to two antiparallel α-helices, which is helical hair pin
(HH).
 In lowermost layer there is WH domain which is most divergent in sequence
amongst the RecQ helicases.

Quaternary structure: RecQ1 has a quaternary structure; it is homodimer. It consists of

two identical subunits (chains A, B), except 1,2-ethanediol (EDO) which presents in chain
B only. The two subunits are designed as head to tail.

Question 4. With the aid of a simple generic diagram, identify and explain how
the type(s) of chemical bonding stabilizes a secondary structure that is present in
your PDB protein. (Up to 50 words)(3 marks)

The catalytic K119 produces a water-mediated connection with Mg2+.Likewise, D219 and
E220 of the DEVH box (motif II) generate water-mediated hydrogen links to the Mg2+.
D379 and ribose O3’ from D2, motif V, are involved in forming a single hydrogen bond
with ADP.

The zinc domain (amino acids 419–480), lies below D2 motif, includes a 4-cysteine zinc-
binding motif, linked to two antiparallel α-helices, which is helical hair pin (HH).

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Question 5. (i) In your PDB protein you were given the sequence position of a
particular amino acid that is mutated to another amino acid. Draw the structure of
the two amino acids. (1 mark)

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(ii) Describe why this position in your protein is important and outline the effects
the mutation will have on the 3D structure and the function of your protein. (up to
50words)(4 marks)
The amino acid 219 is aspartic acid, D219. D219 generates water-mediated hydrogen
link to the Mg2+; which is essential for ATPase activity. Hence, mutating D219 (aspartic
acid), which is negatively charged, to isoleucine, which is non-polar, abolishes the
hydrogen link and eradicates ATPase and helicase activity.

Question 6.
1CJY inhibitor 2V1X inhibitor
O H2N
O N
S S
CF 3 O
H3C O N
OH
OH OH
1IG8 inhibitor 4ZEL inhibitor
OH
S
O OH
O HN N F
HO
NH
HO
H3C F

1ALD inhibitor 1ZOY inhibitor

O OH
P O
O OH OH
HO O
HO
HO
P
O O O Page 8 of 12

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Your enzyme is inhibited by one of the compounds shown in the corresponding

box above. The inhibitor for your specific protein is indicated via the PDB protein
code in the box.

On the basis of the enzyme’s structure, its substrate and mechanism of action,
predict what type of enzyme inhibition(i.e., competitive or non-competitive) may
occur and justify why you think this is the case (Up to 50 words).
(4 marks)

2V1X inhibitor seems to be polar aromatic bulky compound, and therefore this compound
is supposed to bind on the enzyme’s surface away from the active site, altering the
shape of the enzyme so that even if the substrate can bind, the active site functions less
effectively. Hence, this is a non-competitive inhibitor.

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Question 7.Assuming the product formed from your enzyme has an absorbance
at 405 nm, the following data for constructing a standard curve were obtained.

Tube Product Stock Absorbance

[Product] solution* Buffer (405 nm)
(mM) (mL) (mL)
1 0 0 5 0.000
2 10 1 4 0.35
3 15 1.5 3.5 0.65
4 20 2 3 0.83
5 30 3 2 1.31
6 40 4 1 1.73
7 50 5 0 2.03

*The concentration of Product Stock solution: 50 mM.

(i) Calculate the concentration of product [Product] in each tube used to

generate the standard curve. Write down your answer in the table above. (2
marks)
Shown in the table.
(ii)Draw a standard curve using this data set in the grid provided below.(3
marks)

(iii) a) Construct an empty table with the following column headings: Substrate
concentration [S] and initial velocity (Vi) where [S] has the unit µM, and Vi has the
unit mM/s.(2 marks).

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[S] µM Vi (mM/s)
10 21
25 27
50 31
100 36
150 41
200 42.5
Km= 13.5 µM
Vmax= 43 mM/s

(iv) b) Prior to this exam, you were provided with enzyme kinetic data for your
mutated enzyme, whereby Vi was expressed using the unit ∆A(405 nm)/s. Using the
standard curve, express Vi with the unit mM/s rather than ∆A(405 nm)/s. Place your
answer in the table above alongside the appropriate [S].(2 marks)

Hint:To answer this question you need to use the standard curve drawn in
question 7(ii) AND the Vi and [S] data provided prior to the exam.

(v) The unmutated form of your protein has a Km of 25 µM and a Vmax of 43 mM/
s. The enzyme kinetic data for your enzyme with the amino acid substitution
should now be displayed in the table above. Based on these data, explain the
effect of amino acid substitution on the Km and Vmax for the mutated protein.(Up to
50 words) (5 marks)

Hint:You are not expected to draw a Lineweaver-Burk plot, however a quick

sketch of a Vi vs [S] plot may help you to answer the question (note: no marks
will be assigned to this graph and it does not have to be included in your
response to this question).

My estimated values are Km= 13.5 µM, Vmax= 43 mM/s

We can see that Km value decreased dramatically, while Vmax value remains the
same (unchanged).

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Question 8. A transmembrane protein uses a β barrel structure to span the cell

membrane.How can amino acid mutations in a protein within this β barrel
structure affect interactions with the membrane and why? (Up to 50 words) (4
marks)

β-barrel structure in a transmembrane protein is made of β-sheets of non-polar

amino acids connected by linker peptides that are made of polar amino acids.
β-barrel structure is made of non-polar amino acids because they have to interact
with the hydrophobic lipid bilayer. Any change in the amino acid sequence (by
mutations) of β-barrel structure would affect this interaction. Especially, if any
non-polar amino acid is replaced by polar amino acid, then the interaction
between β-barrel structure and the membrane would be heavily disturbed.

Question 9. Haemoglobin is a protein that carries oxygen in our red blood cells.
Sickle cell anaemia is a genetic disease in which Glu at position 6 of
haemoglobinis mutated to a Val, rendering haemoglobin non-functional.

(i) What type of amino acid change is this? (1 mark)

Sickle cell anemia is an example of a point mutation where a Glutamic acid (acidic amino
acid) changes to a Valine (Nonpolar amino acid). From a polar and acidic amino acid
(Glu) to a nonpolar and hydrophobic amino acid (Val). The change of the amino acid Glu
to Val changes the structure of the hemoglobin, forming a sickled structure.

(ii) Give one reason why this could affect the function of this protein.(1 mark)

One possible reason that affect the function of the protein is the difference in the polarity
of the two amino acids. The Glutamic Acid is a polar acidic amino acid and the Valine is a
hydrophobic amino acid. The change in amino acid results to a change in the polarity of
the protein that can affect the structure. For a quaternary protein such as Hemoglobin,
Hydrophobic interaction is one factor for the structure of the protein. The change of Glu
to Val causes a change in the structure of the protein that causes to form a sickled
structure. A sickled structure of the hemoglobin will result to a lower capacity to carry
oxygen in the body.

Question 10.Lectin is a protein that binds carbohydrates and is critical for

biological recognition processes in living organisms. Describe what bonds and
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forces could be involved in lectin and carbohydrate interactions. (Up to 50 words)

(2 marks)

Protein (lectins) and carbohydrates interact via hydrogen bonds and van der Waals force.
In protein (lectins)-carbohydrate interaction, hydrogen bonds bridge hydroxyl groups of
monosaccharides and the amino groups of amino acids. Van der Waals forces connect
hydrophobic sugar faces with aromatic amino acid side-chains.

Question 11. A hexokinase is an enzyme that adds a phosphate to glucose after it

enters the cell, which is considered the first step of glycolysis. One enzyme,
hexokinase A (HKA), has a Km of 0.02 mM, whereas another, hexokinase B
(HKB), has a Km of 1.0 mM. Explain why some types of fast growingcancer cells
would use HKA instead of HKB. (Up to 50 words)(4 marks)

Cancer cells favour hexokinase A (HKA) over HKB because it has low KM than HKB.

Cancer cells favour hexokinase A (HKA) over HKB because it has low KM than HKB.
Low KM means high-substrate affinity. HKA is able to achieve half the velocity at 0.02
mM, while HKB requires more substrates (1 mM).
This means that HKA has high affinity to glucose than HKB does.
This is why cancer cells favour HKA over HKB.

Question 12. The cell membrane is a structure composed of phospholipids.

Explain why phospholipids arrange to form a bilayer. Would you expect the
molecule shown below to form a bilayer in water– why or why not? (Up to 50
words)(4Marks)
O

H3C OH

Phospholipids arrange to form bilayers because the hydrophobic regions find ways to
remove themselves from water while the hydrophilic regions interact with water and a
layer called a lipid layer is formed. The molecule shown would form a bilayer in water
because it is non polar. Hydropholic head of the phospholipid molecules dissolve readily
in water while the long fatty acid chains of phospholipids are non polar hence they avoid
water because of their insolubility.
The ends of non polar molecules are not charge.
The molecule shown are non polar and water is polar hence it cannot dissolve in water.
phospholipid molecules and water molecules do not bond or share electrons in any way.

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Question 13. Based on your knowledge of the chemical structure of DNA, explain
why all DNA molecules move in the same direction towards the positive electrode
when placed in an electric field. Name one analysis toolin biotechnology which
uses this feature.(2 marks)

DNA (and RNA) has net negative charge as it has phosphate groups in its
phosphodiester bonds. These phosphate groups contribute negative charge to
nucleic acids. That is why DNA moves toward positive electrode.
This property is used in the agarose (or polyacrylamide) gel electrophoresis of
DNA separation where DNA samples are always loaded on the negative
electrode side of the gel. Upon applying electric field, the DNA would then move
towards positive electrode.

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