Proteins, Enzymes, and Metabolism

This assignment is for students in the Faculty of Science, Engineering and Computing, Department of Biomolecular Sciences and Applied & Human Sciences. The assignment is divided into three questions: Enzymes, Protein Structure, and Protein Purification. The first question focuses on enzymes, discussing their importance, properties, and regulation. The second question covers protein structure, including topics such as hydrogen bonding in alpha-helix and beta-sheets, tertiary structure formation, and the interaction between FMN and azoreductase. The third question is about protein purification, covering topics such as exploiting protein properties for purification, breaking open bacterial cells, preserving biological integrity, calculating specific activity, fold purification, and percent recovery, and explaining the use of SDS electrophoresis to deduce purity.

Faculty of Science, Engineering and Computing
Departments of Biomolecular Sciences and Applied &
Human Sciences
LS5002 Proteins and Metabolism
Enzymology, Protein Structure and Protein Purification
Workbook
Instructions to Candidates
This paper contains THREE QUESTIONS
These questions are designed to parallel the teaching delivered in Teaching Block 1,
weeks 1 to 9. This workbook represents the major assessment of the material
(Enzymes, Protein Structure and Protein Purification), which will not be covered by
the end of module examination.
Submission:
Deadline: 23:59 on Thursday 6th December 2018
Submission of the completed workbook will be to an electronic submission box on
Canvas. The submitted script should be a single Word document with any figures
embedded into the document.

Question 1: Enzymes
(a) Why are enzymes vital for living organisms? Include in your answer four
properties of enzymes to illustrate your answer. (3 marks)
Enzymes are biological catalysts that are essential for speeding up and activating
biological reactions within living organisms, where without the enzyme may have
taken a long time to occur or may not have occurred at all. Enzymes catalytic power
is greater than any existing chemical catalysts, resulting into tremendous increases
in reaction rates. They function in aqueous environment, which is crucial in the
environment of a living organism with mild pH and temperatures. Enzymes can be
regulated by other molecules usually through the process of inhibition, which
changes the active site to activate or deactivate the enzyme; and because they are
highly specific, the inhibitors need to be as well avoiding any inhibition to different
enzymes.
(b) “Enzymes are 100% protein.” Explain why this statement is incorrect,
illustrating your answer with two named enzymes and their respective non-
protein components.
(3 marks)
Not all enzymes are protein, a few commonly known exceptions are catalytic RNA’s
(ribozymes). Ribozymes are ribonucleotides, which are made up of ribose sugar, a
phosphate group and a nitrogenous base, where as proteins are made up of amino
acids.
(c) Describe, in detail, how enzymes decrease the activation energy for a
reaction, and how this also relates to the specificity of the enzyme. (5
marks)
Enzymes
(d) In developing their mathematical model for invertase, what assumptions were
made by Michaelis & Menten to derive their model? (4 marks)
(e) Explain the meaning of Km and Vmax, in the context of the Michaelis-Menten
model. (2 marks)
Km is the concentration of substrate that is required to reach half of Vmax, where
Vmax is the maximum rate of reaction.
(f) Outline the mechanism of catalysis of chymotrypsin, with reference to the
amino acid residues involved (5 marks)

Total = 22 marks
Question 2: Protein Structure
(a) Give an example of an amino acid whose side chain has two atoms able to
form hydrogen bonds. Include a diagram to support your answer. (2
mark)
(b) Compare and contrast hydrogen bonding in α-helix and β-sheets. (5 marks)
(c) Describe the interactions which lead to the formation of tertiary structure.
(5 marks)
(d) Produce a figure clearly showing all interactions between FMN and the
azoreductase from Klebsiella pneumoniae. Ensure to show all atoms which
interact with FMN and label all residues of which they are part. Remember
that both peptide backbone as well as side chain residues can form hydrogen
bonds. Ensure to clearly differentiate between residues from different
monomers, also colouring carbon atoms of ligands a different colour to the
surrounding peptide so that they stand out, State the PDB identifier for the
structure used to generate the image. (8 marks)
Total = 20 marks

Question 3: Protein Purification
(a) List four properties of proteins that could be exploited in their purification
(2 marks)
(b) Give two procedures that can be used to break open bacterial cells? In your
answer briefly state the principle behind how each method achieves this.
(4 marks)
(c) What conditions/reagents can be used to preserve the biological integrity of
proteins once cells have been broken open? You must provide at least four
examples and explain how the condition/reagent works to preserve the
biological integrity. (4 marks)
(d) Use the table below to calculate the specific activity, fold purification and
percent recovery for the last three stages of a purification procedure. Please
show at least one full calculation for each column and where appropriate
included the correct units. (3 marks)
Purification
Procedure
Total
protein
(mg)
Total
Enzyme
Activity
(units)
Specific
Activity
Fold
Purification
Percent
Recovery
(%)
Crude extract 5,000 200,000 40 1 100
Ammonium
sulphate 2,000 150,000
CM-cellulose
chromatograph
y (pH6.5) 50 125,000
Gel permeation
(exclusion)
chromatograph
y
10 80,000

(e) Explain what a crude extract is and why ammonium sulphate was used as the
first step in the purification procedure. (3 marks)
(f) Why was CM-cellulose ion exchange chromatography chosen for the
purification of an enzyme with a pI of 8.9, when using a buffer with pH 6.5?
(2 marks)
(g) A small sample of the purified enzyme was run on SDS electrophoresis gel.
Explain briefly the technique of SDS electrophoresis and how it can be used
to deduce the purity of the bacterial enzyme and confirm that this enzyme
consists of two subunits. (4
marks)
Total = 22 marks