e usP

THE UNIVERSITY OF THE

SOUTH PACIFIC

CH405

BIOCHEMISTRY

FSTE
School of Biological and Chemical Sciences

Final Examination
Semester 2, 2018

Face to Face Mode

Duration of Exam: 3 hours + 10 minutes

Reading Time: 10 minutes
Writing Time: 3 hours

INSTRUCTIONS

This exam has SIX questions.

Candidates are to answer FIVE in the answer book provided.
Each question is worth 20 marks.
Answer each question on a NEW page.
This exam is worth 40% of your overall mark.

Materials
You may use a non-programmable calculator.
You will be provided with a graph paper.
Question 1

a) A nonapeptide (a 9-amino acid peptide) was purified from the pituitary gland and was
predicted to be oxytocin, a hormone and a neuropeptide. To confirm the nonapeptide to be
oxytocin, a biochemist carried out the analysis and the results are given as follows:

 One cycle of Edman degradation of nonapeptide followed by treatment with 2-

mercaptoethanol yields 1 mol of PTH-cysteine (Cys) per mole of the peptide.

 The nonapeptide was hydrolysed by chymotrypsin which yields two peptides with the
composition (Cys, Ile, Tyr) and (Met, Cys, Gly, Gln, Pro, Leu).

 One cycle of Edman degradation of fragment (Met, Cys, Gly, Gln, Pro, Leu) yields 1 mol of
PTH-glutamine (Gln) per mole of the hexapeptide fragment.

 Treatment of 1 mol of nonapeptide with two cycles of carboxypeptidase yields 1 mol of

glycine (Gly) followed by 1 mol of leucine (Leu).

 Treatment of the intact pentapeptide (Met, Cys, Pro) with CNBr yields two peptides with
the composition (homoserine lactone), while the intact peptide (Met, Cys, Pro) yields PTH-
cysteine in the cycle of Edman degradation cycle.

Note:
 PTH-amino acid: phenylthiohydantoin derivative of the N-terminal amino acid.
 Chymotrypsin cleaves at Tyr and Phe residues at the C-terminus of the peptide bond.
 Homoserine lactone is formed when CNBr reacts with Met residues.
 Amino acids are separated by commas when their sequence is not known.

Determine the sequence of this nonapeptide from the information above and compare it
to the structure of oxytocin given below.

~---s-s---~
Oxytocin - ~ - fy-1~-~-~n-~-~-~-G~
(Mammals) 1 2 3 .4 5 6 ~ 8 9

(10 marks)

2
b) Protein folding is governed by a number of interactions and forces. Briefly explain the process
involved in protein folding.
(5 marks)

c) Protein misfolding is a common event in living cells. Explain the role of molecular chaperones
in the event of misfolded proteins.
(5 marks)

Question 2

a) Given below is the crystal structure of pyruvate kinase from rabbit (A) and human (B).

A B
Demina, A., et al, 1998, Blood, 92:647- https://cansar.icr.ac.uk/cansar/molecular-
652 targets/P00549/
Also known as KPYK1_YEAST, CDC19, PYK1

i) Identify the main structural features in each of the 3 domains in the pyruvate kinase
derived from rabbit (A)?
(3 marks)

ii) Select TWO of the structural features identified in (a) above and explain how these
features are formed/held in the structure of pyruvate kinase?
(2 marks)

3
 iii) Comment of the conserved domains in pyruvate kinase from rabbit (A) and human (B).
(3 marks)

b) Explain the characteristic hydrogen bonding pattern in:

 An α-helix
 A beta Sheet
 A collagen triple helix
(3 marks)
c) The larval form of the parasite Schistosoma mansoni infects humans by penetrating the skin.
The larva secretes enzymes that catalyze the cleavage of peptide bonds between amino acids X
and Y in the sequence –Gly-Pro-X-Y- (X and Y can be any amino acid). Suggest a possible
reason for this enzyme activity for the parasite.
(3 marks)

d) Electron transport chain is the last step in carbohydrate metabolism. Briefly explain the
following in regards electron transport chain:

i) Significance of the reaction

ii) Energetics and efficiency of energy production.
iii) Regulation of electron transport chain.

(6 marks)

Question 3

a) Describe the main biochemical processes involved in fatty acid biosynthesis. You may want to
use the following:
i) Synthesis of malonyl acyl carrier protein and acetyl acyl carrier protein
ii) Initiation of fatty acid synthesis
iii) Elongation reactions
iv) Fatty acid activation
v) Fatty acid extension and desaturation
vi) Possible role of immune cell in fat metabolism
vii) Fat metabolism in obese individual
(14 marks)
b) Discuss the fates of Acetyl Coenzyme A in the biological system.
(6 marks)

4
Question 4

Consider the oxygen carrier globular proteins - myoglobin and hemoglobin.

a) Comment on the structure of these two globular proteins.

(5 marks)

b) Identify the critical amino acids near the heme group of hemoglobin amino acids and explain its
importance in terms of hemoglobin function.
(5 marks)

c) Discuss the binding of oxygen to the heme group and how this binding affects the structure of
the protein.
(5 marks)

d) There are two possible ways oxygen binding to the heme group is being controlled. Identify
ONE of the two systems and explain briefly how the oxygen binding to the heme group is being
regulated.
(5 marks)

Question 5

Given below is a schematic representation of Renin-Angiotensin system.

5
One of the key enzymes that regulate the blood pressure is Angiotensin Converting Enzyme (ACE)
which inactivates a nonapeptide bradykinin by hydrolysing the carboxylic end of the 8th amino
acid.
i) Explain the role of Bradykinin in the vascular system.
(3 marks)

ii) Given below is the amino acid sequence of bradykinin. What other possible site would ACE
able to further hydrolyse bradykinin? Explain your answer.

Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9
(2 marks)

iii) ACE is a zinc metallopeptidase. What could possibly be the role of Zn metal in the activity
of ACE?
(2 marks)

iv) Given below are two inhibitors of ACE. Give a brief structural analysis of these inhibitors
with bradykinin structure.

Captopril

Enalapril

(4 marks)

v) Inhibitors can either have reversible or irreversible binding properties. Explain the effect of
reversible and irreversible inhibitors on enzymes.
(5 marks)

vi) Aminopeptidase P (APP) is another enzyme that inactivates bradykinin by hydrolysing the
carboxylic end of the proline (Pro), which is the 2nd and 3rd amino acid in the sequence.
Design TWO peptide based inhibitors of APP based on this information.
(4 marks)

6
Question 6

Alkaline phosphatase catalyzes the hydrolysis of p‐nitrophenylphosphate to para-nitrophenol at pH 8

and 37°C.
OH
O=~- ~NO
I v ~- 2
+ H20
Alkaline
Phosphatase
HO
-0-" _ \; N02 + H3P04
OH

p-nitrophenyl phosphate p -nitrophenol

The kinetic data in the following table were obtained by monitoring the formation of the para-
nitrophenol at 240 nm (A240) with and without inhibitor of alkaline phosphatase.

Substrate Initial velocity µM/sec Initial velocity µM/sec

mmol/L) [I] Inhibitor=0 (mM) [I] inhibitor= 5 mM
5 2.73 1.82
10 5.00 3.33
20 8.57 5.71
50 15.00 10.00
Note: Enzyme concentration is 0.0015 μM

i) Use this data to sketch a Line-weaver Burk Plot.

(10 marks)
ii) Determine the maximum velocity (Vmax) with and without inhibitor.
(2 marks)

iii) Determine the Km values with inhibitor and without inhibitor. Explain what Km value means.

(3 marks)

iv) Using the values from ii) and iii) above, determine if the inhibitor as either competitive or
uncompetitive or non-competitive. Explain your answer.
(2 marks)

v) Explain how pH and temperature affect the activity of alkaline phosphatase.

(3 marks)

*********************************The End**********************************