SLE212 Biochemistry T1 2019 Assessment Worksheet C

Worksheet C
Enzymes
Name
Prac Partners
Prac date
Due date

1) Electronic Format
The worksheet must be produced entirely in an electronic fashion, however, we do not accept ePens! Hand-
written or hand-drawn components, be it as original or as scanned image, will not be accepted/not receive any
marks! Submit electronically as either a Word or PDF file via the designated CloudDeakin dropbox.

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2) Plagiarism and Collusion
Before submitting your worksheet to the dropbox submit it to the Check Your Work: Turnitin Assignment Folder

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available on CloudDeakin. In case that we find that work is the result of plagiarism (from foreign source like

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internet or fellow student) or collusion we report this to the Faculty Academic Progress and Discipline Committee.

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Note: It is NOT sufficient to submit your worksheet only to the Turnitin Folder! Your worksheet will be marked only
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if you submit it to the appropriate Dropbox!
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3) Due dates for worksheet submission:
Worksheet A is due 5.00 pm two teaching weeks after practical session 1, this is the day BEFORE your prac 2
Worksheet B is due 5.00 pm two teaching weeks after practical session 2, this is the day BEFORE your prac 3
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Worksheet C is due 5.00 pm two teaching weeks after practical session 4 (!), this is the day BEFORE your prac 5
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Worksheet D is due 5.00 pm ONE (!) teaching week after practical session 5 (!)
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4) Referencing
All foreign sources of information, i.e. information that was not provided in this unit, must be
cited. The citation style to be used is Harvard style, for more information about this style and referencing in
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general see: https://www.deakin.edu.au/students/studying/study-support/referencing. Citations will be

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considered only when they are placed as in-text citations such that it is clear which statement they do support.
Furthermore, bibliographic details of a citation must be listed within the question where they are used, reference
lists at the end of the worksheet will not be accepted! The citation needs to be a quality journal article, book or
refereed website. No marks are awarded if Wikipedia or poor-quality websites (eg. the website of a course at
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another institution) are cited.

The practical manual, the prac script, OLMs and lecture notes can NOT be cited! Citations of text
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books are often most appropriate for the level of this unit and therefore encouraged! If you do cite a
textbook you MUST indicate relevant page numbers!

5) Learning Outcomes:
Q1,2,4,7,8: Discipline specific knowledge and understanding.
Q3,5: Written communication. Presentation of experimental data.
Q6: Quantitation skills. Application of mathematical equation and discipline specific knowledge to
quantitatively describe a system.
Q6: Critical thinking & problem solving. Use of discipline specific knowledge and logic to predict the
chemical behaviour of a biochemical compound.

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 SLE212 Biochemistry T1 2019 Assessment Worksheet C

Please note: this worksheet covers practicals 3 and 4 to equal parts,

i.e. for both practicals you can obtain a maximum of 4.5 marks.
Questions related to practical 3:

1. Research the enzyme Bromelain, EC 3.4.22.33.

Indicate i) the class of enzymes to which it belongs to, ii) name the kind of bond that is modified due to
its activity, iii) the degree of substrate specificity and iv) how it affects your experience when eating
pineapple. Must be less than 50 words total (1 mark)

i) Bromelaine breaks down protein by a hydrolysis reaction. Therefore it is classified as a

protease (Maurer, 2001; Hetano et al., 1995) 2)
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ii) The bonds modified due to its activity are peptide bonds.

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iii) Substrate specificity is selective.

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iv) Bromein is naturally found in pinaple. When consumed it starts to break down the proteins
in the mouth causing it to digest the tender skin, resulting in discomfort and sometimes pain
(Maurer, 2001).
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Hatano, K., Kojima, M., Tanokura, M. and Takahashi, K. (1995). Primary Structure, Sequence-
Specific 1H-NMR Assignments and Secondary Structure in Solution of Bromelain Inhibitor VI
from Pineapple Stem. European Journal of Biochemistry, 232(2), pp.335-343.
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Maurer, H. (2001). Bromelain: biochemistry, pharmacology and medical use. Cellular and
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Molecular Life Sciences, 58(9), pp.1234-1245.

your experiments you used acid phosphatase to hydrolyse the substrate p-nitrophenol phosphate. i)
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comment on the biological relevance of this reaction and ii) what this reaction teaches us about the
substrate specificity of the enzyme. Must be less than 50 words total. (0.5 marks)
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i) For life to occur as we know it, most living organisms require to hydrolyse substrates by
means of enzyme catalytic activity similarly to the hydrolisation of p-nitrophenol by acid
phosphatase observed in the experiment.

ii) This reaction teaches us that enzymes have can have different affinities to substrates and
that factors such as temperature and PH can have an impact on enzyme affinity.

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 SLE212 Biochemistry T1 2019 Assessment Worksheet C

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 SLE212 Biochemistry T1 2019 Assessment Worksheet C

3a) Using data from prac 3, produce a categorical bar graph of the effect of temperature on acid
phosphatase activity using Excel. Include a suitable title, labelling of the axes of the graph and a
figure legend. (1 mark)

Figure 1. Categorical graph illustrating the enzyme activity of acid phosphatase

in different temperatures (4˚, 25˚, 37˚, 55˚ and 70˚). Enzymatic was measured by
absorbance of 405 nm. The trend of the graph shows a continuous increase in
enzymatic activity up to 55˚ followed by an abrupt decrease as the temperature
was increased to 70˚.

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3b) In 100 words or less write a short conclusion about the effect of temperature on enzyme activity.
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Include in your answer the trend observed and explain this trend using your biochemical knowledge of
enzymes and reactions. (1 mark)
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As described by the graph above, temperature had a major impact on enzyme activity.
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Enzyme activity increased significantly up to 55˚ due to an increase in kinetic energy in the
substrate as a consequence of tempearature rise. However, after a temperature of 70˚ was
reached which is much higer than the ideal temperature for enzyme activity, enzymes began
to denature which is caused by the break down or destruction of the protein structure
causing a change in the shape of the enzyme and affinity.

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 SLE212 Biochemistry T1 2019 Assessment Worksheet C

4) Alcohol dehydrogenase is involved in the metabolism of alcohol. A cytoplasmic version of the

enzyme catalyses the conversion of alcohol to acetaldehyde and a mitochondrial version catalyses
the conversion of acetaldehyde to acetate, see reactions below.

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Some people react to the consumption of alcohol with the appearance of a facial flush.
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In less than 100 words total i) name the biochemical basis for this physiological reaction and ii)
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explain in the language of this unit how the enzymatic properties of alcohol dehydrogenase relate to
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this physiological reaction. (1 mark)

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i) there is a build up of acetaldehyde not broken down by the mitochondrial version of

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alcohol dehydranase.
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ii) The mitochondrial version of alcohol dehydranase is mutated and therefore can reduce
enzyme concentration or affect the tertiary structure of the enzyme. This results in a build up
of acetaldehyde and hence the previous mentioned physiological reaction.

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 SLE212 Biochemistry T1 2019 Assessment Worksheet C

Questions related to practical 4:

5. Produce a Lineweaver-Burk Plot for acid phosphatase activity in the presence and in the absence
of phosphate using Excel. Include a suitable title, labelling of the axes of the graph and a figure
legend. The format for the Lineweaver Burk plot should be similar to that detailed in OLM4 Enzymes
and in figure 6, page 183 of your textbook. Clearly identify the lines that describe data for the
absence and presence of inhibitor and provide in the graph the straight line equations. Identify the
type of inhibitor in the graph. (2 marks)

The colour of the graph lines MUST be RED (you will receive 0 marks for question 4 in case that the
line has a different colour).
Explanatory note: The teaching team strictly enforces the above indicated color requirements in order to ensure the
originality of 2019 worksheet submissions.

Acid phosphotase activity (nm) in the precense and abcense of phophate

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1/ acid phosphatase activity (nm)

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1.5 f(x) = − 0.34 x + 2.2

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-0.1f(x) = − 00.9
x + 0.09 1.9 2.9 3.9 4.9 5.9 6.9 7.9
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-1.5
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-2.5
1/[p-nitrophenol phosphate] (mM/L)
Phophate
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present Linear () Linear ()

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Figure 2. Illustrates the enzamitic activity of acid protease in the precense of phosphate
as described by the legend. Enzymic activity is measured by absorbance of 405 nm light
to calalyse p-nitrophenol phosphate (mM/L).
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 SLE212 Biochemistry T1 2019 Assessment Worksheet C

6. From your Lineweaver-Burk plot, derive the values for the following table. Indicate the appropriate
units! (1 mark)

Km Vmax

Uninhibited 1.098 mM 0.584 mM/min

Inhibited 2.204 mM 0.182 mM/min

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7. In your acid phosphatase assay that you performed in practical 4 in the absence of phosphate, what

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would happen to the Vmax of the enzyme if you were to reduce the substrate concentration by 50%? ii)

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provide reasoning. Less than 50 words total. (1 mark)

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i) A reduction of 50% in the substrate concentration would not cause a change in the V max.
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ii) Vmax is affected by enzyme concentration rather than substrate concentration as the
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amount of ezyme affects the turnover given that the substrate is sufficient.
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8. A final step in the analysis of enzymatic activity during practials 3 and 4 was the addition of NaOH to
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your samples. In less than 100 words provide two reasons why you added NaOH explaining the
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molecular effects that NaOH had in each case! (0.5 mark)

Reason 1) It converts P- nitrophenol to p-nitrophenolate which results in a yellow colour. The

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change in colour from transparent is needed when analysing absorbance.

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Reason 2) It abruptly stops enzyme anctivity. This truncation in the reaction allow for
accurate calculations by controlling the time of reaction. By adding NaOH, the pH increases
and denatures acid phosphatase by creating more alkaline conditions (not optimal for acidic
enzyme activity), hence stopping it from interacting with the substrate.

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