BIOL2565 Cellular Pathways
BIOL2565 Cellular Pathways
Practical reports 3 Enzyme Kinetics
Muna Mohamed , Muneeba Shahzad and Veronique Frere
Carboxylesterases are a multi-gene family of enzymes that catalyze the hydrolysis of esters, amides, thioester and carbamates (Laizure et al., 2013). Carboxylesterase belongs to the esterase group of proteins where their primary function is to break down carboxylic acid esters into the corresponding carboxylic acid and alcohol molecules via a proton transfer hydrolysis mechanism.
Carboxylesterases (CEs) are members of the esterase class of proteins (EC 3.1.1.1), that cleave carboxylic acid esters (RCOOR) into the corresponding carboxylic acid (RCOOH) and alcohol (ROH), via a proton transfer hydrolysis mechanism using a catalytic serine present within a Ser-His-Glu triad. These enzymes are widely distributed in nature and especially common in mammalian liver (Figure 3.1). Mammalian CEs hydrolyse esters, thioesters, and amide-ester linkages of a broad spectrum of structurally diverse compounds. Some of their substrates include clinical drugs such as lovastatin and the anaesthetic lidocaine, anticancer drugs such as capecitabine, the antibiotics Ceftin and Vantin, and angiotensin-converting enzyme inhibitors delapril, imidapril, and temocapril. These enzymes can also cleave narcotics such as heroin and cocaine, as well as toxic chemical agents such as sarin, soman, and tabun, resulting in their detoxification. However, if the dose taken by an individual is high, the enzyme system gets overwhelmed, and an individual may die as a result due the toxic nature of some of these compounds. CEs also play an important role in cholesterol and fatty acid metabolism. Carboxylesterases are inactivated by the phosphate diester, bis (p-nitrophenyl) phosphate, which is non-toxic (Figure 3.2).
Carboxylesterases (CEs) belong to the esterase group of proteins (EC 3.1.1.1). Their primary function is to break down carboxylic acid esters (RCOOR) into the corresponding carboxylic acid (RCOOH) and alcohol (ROH) molecules. This process occurs through a proton transfer hydrolysis mechanism, facilitated by a catalytic serine within a Ser-His-Glu triad. These enzymes are widespread in nature, particularly abundant in mammalian liver tissues.
Mammalian CEs exhibit a broad substrate specificity, capable of hydrolyzing esters, thioesters, and amide-ester linkages found in a diverse range of compounds. Among their substrates are various clinical drugs like lovastatin, lidocaine, capecitabine, Ceftin, and Vantin, as well as certain anticancer agents and angiotensin-converting enzyme inhibitors. Additionally, they can metabolize narcotics such as heroin and cocaine, along with toxic chemicals like sarin, soman, and tabun, thereby facilitating detoxification.
However, when exposed to high doses of these compounds, the enzyme system may become overwhelmed, potentially leading to fatal consequences due to the toxic nature of some substrates. Beyond their detoxification role, CEs also play a significant part in cholesterol and fatty acid metabolism.
Interestingly, carboxylesterases can be rendered inactive by phosphate diesters like bis(p-nitrophenyl) phosphate, which is non-toxic. This characteristic is illustrated in Figure 3.2
5. Introduction, this section should be brief, but contain the background information necessary to understand the purpose of the practical. It should be written by you rather than copied from the introduction / background section of the practical notes, because it is your experience of the practical you are writing about, which may often be different to the intended outcomes, and coping from the practical notes is plagiarism, and this is not allowed at University.
You may also need to seek out other information on the topic of the practical to ensure you have the background required for the reader to understand the aims of the practical. If so, make sure you reference all sources correctly. Please go the following site http://www1.rmit.edu.au/library/referencing-guides for more information.
Do not write more than 0.5-1 page on the Introduction, it should be brief and relate to what the prac is about. If you are not sure then look at some scientific papers as the Introduction for the prac report will be along similar lines to the Introduction for a paper (though shorter in length and in detail).
Materials:
Solution A 0.01 M Tris buffer [2-amino-2-(hydroxymethyl)-1,3-propanediol}, pH 8.0
Solution B 2mM p-nitrophenyl acetate (p-npa)
Solution C 0.035 mg/mL Carboxylesterase in 0.01 M Tris buffer, pH 7.0
Solution D 2.5 mM butyraldehyde in 0.01 M Tris buffer, pH 8.0
Tube rack
3mL plastic cuvettes x 18
Parafilm
Part 1: Effect of Enzyme Concentration on reaction kinetics
Place six of the 3mL plastic cuvettes into a tube rack
Set up the six cuvettes as displayed on the table 1.1. Please note that you will set up duplicate blanks (tube 1).
Add only solutions A and B to each cuvette with the corresponding volumes. Adding Solution C (enzyme) as instructed in Step 7 as it will initiate a reaction.
Table 1.1. Protocol for measuring the effect of enzyme concentration on reaction kinetics
Reagent Tube 1A Tube 1B Tube 2 Tube 3 Tube 4 Tube 5
0.01 M Tris buffer, pH 8.0 2.800 2.800 2.790 2.780 2.750 2.700
2 mM p-nitrophenyl acetate 0.200 0.200 0.200 0.200 0.200 0.200
0.035 mg/mL carboxylesterase 0 0 0.010 0.020 0.050 0.100
Move to a spectrophotometer and set it up to perform a Time scan. Set the wavelength at 400nm, total scan time is 120 seconds, and the time interval is 1 second.
Place the first Tube 1A in the reference (back) compartment of the spectrophotometer. Similarly, place the Tube 1B into the sample compartment (front). Autozero the spectrophotometer. Once you have done this, you can remove the cuvette (Tube 1B) from the sample (front) compartment.
Next add the cuvette from Tube 2 and add the 0.01mL of the enzyme (Solution C) to the cuvette. Place a parafilm over the top off the cuvette and invert the mixture to mix the enzymes and initiate the reaction. Place the cuvette back into the sample compartments and select start to begin the scan.
When the time scan is complete, collect the printout of the scan and the data list.
Repeat steps 6-7 for all the other tubes/cuvettes (Tubes 3-5) using Tube 1A as the blank.
Part 2: Effect of the inhibitor butyraldehyde on enzyme activity
Place the corresponding volumes of solution A and B into the plastics cuvette as seen on Table 1.2. The addition of Solution C (enzyme) will start the reaction, so do not add this to your cuvettes until instructed to do so in step 4.
Table 1.2. Protocol for measuring the effect of substrate concentration has on enzyme activity in the absence of inhibitor.
Reagent (mL) Tube 1A Tube 1B Tube 2 Tube 3 Tube 4 Tube 6
0.01 M Tris buffer, pH 8.0 2.950 2.950 2.940 2.930 2.900 2.850
2mM p-nitrophenyl acetate 0 0 0.010 0.020 0.050 0.100
0.035 mg/mL carboxylesterase 0.050 0.050 0.050 0.050 0.050 0.050
Set up the spectrophotometer to perform a 2 min Time scan. Set the wavelength at 400nm. Total scan time = 120 seconds, and the time interval is 1 second.
Place the contents of Tube 1A into a cuvette and place it in the reference (back) compartment of the spectrophotometer. Similarly, place the contents of Tube 1B into a cuvette and place it in the sample compartment (front). Autozero the spectrophotometer.
Add 0.050 mL of the enzyme to both cuvettes, using parafilm, invert the cuvettes and place in the holder. Close the cover and select start to begin the 2-minute scan.
Remove cuvette 1B from the spectrophotometer, leaving the blank cuvette (Tube 1A) in the reference (back) compartment of the spectrophotometer. Place Tube 2 into the sample compartment (front). Autozero the spectrophotometer.
Add 0.050 mL of Solution C (enzyme) to Tube 2 cuvette. Place a piece of parafilm over the top and carefully invert to mix the enzymes. Reinsert the cuvette back into the sample compartment.
Close the cover and select Start to begin the 2-minute scan. When completed, remove the cuvette from the sample compartment and collect the printouts.
Repeat steps 5-7 for the remaining Tubes 3-5, using Tube 1 as your blank.
Now set up another set of 5 Tubes to determine the effect of 2.5 mM butyraldehyde on enzyme activity. Using Solution D to set up the tubes. Using the corresponding volumes shown on Table 1.3., repeat the steps listed above from 2-8.
Table 1.3. Protocol for measuring the effect of 2.5 mM butyraldehyde has on enzyme activity.
Reagent (mL) Tube 1A Tube 1B Tube 2 Tube 3 Tube 4 Tube 5
2.5 mM butyraldehyde in 0.1 M Tris buffer, pH 8.0 2.950 2.950 2.940 2.930 2.900 2.850
(B) 2 mM p-nitrophenyl acetate 0 0 0.010 0.020 0.050 0.100
0.035 mg/mL carboxylesterase 0.050 0.050 0.050 0.050 0.050 0.050
7. Results, this is the important section of the report and includes all the results obtained from the prac. In your report use single line spacing and a minimum 12 point font size. All Figures and Tables must have a title, a figure legend and labelled axes with the units specified. There must also be a written results section describing the results and making reference to all figures and tables (eg see Fig. 1). If abbreviations are to be used, ensure they are written in full at the first occurrence, with the abbreviation (abbrev.) in parenthesis. Figures, tables and text should be designed to clearly and concisely show the reader what happened in the experiments.
Please note: You do not start the results section with a Table or a Figure you need to write some text describing what you did first and then refer the reader to the results that are shown in the Table or Figure.
References should be cited at the end of the document and acknowledged in the text. The use of appropriate referencing, to books, journal articles and online sources, is an effective way of reducing claims of plagiarism (1).
Please see an example of a good and bad figure below in Fig 1. Graphs must be correctly labelled on both the X and Y axes, there must be a legend and a title, and the correct units mentioned.
Figure 1: Shows the effect of three drug treatments on some cells. The axes on the left figure are poorly labelled and would not receive full marks and the graphs are not very clear. The figure on the right hand side gives the reader more information. Always remember to refer to a figure in the text, a figure that is not referred to will cost you marks.
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If you are preparing a line graph showing X vs Y, then you should make sure it is drawn properly. Using Excel is not the best way to draw a graph, and you would be advised to use this software package to draw graphs (see http://www.excel-easy.com/examples/line-chart.html or https://www.youtube.com/watch?v=Rn_275psJFc)
Tables all should be labelled correctly and the data set out in rows or columns. Units must be correctly mentioned. The table should have a title and reference to it must be made in the text of the report, similar to that for the Figures.
If you have had to calculate data you need to show a sample calculation in your report, to show the reader that your data manipulation is correct. You only need to show one sample calculation for each set of data, the rest of the modified data is shown summarized in a Table. These sample calculations should appear before the related Table or Figure which has all the data in it.
Remember: In all the pracs in this course there are calculations, so you only need to show one calculation for the data that appears in that Table or Figure. You may need to show more than one set of calculations in your report.
If you include images in your report, you will need to label them as Fig X, and give them a label as well as a short description of what it is about. The purpose of a label is that briefly informs the reader what the figure is about.
8. Discussion, in this section the results should be discussed in relation to the aims or hypothesis of the study and whether or not there are potential confounding factors due to methodological limitations or errors. If you read other sources of information, make sure you reference them. Practical reports are notorious for raising claims of plagiarism, read it, quote it or rewrite it from scratch in your own words and reference it. Do not copy someone elses work as this is plagiarism and you will receive a 0 for the report if you do so, and you will be asked to explain yourself to the Head of School in regards such matters.
There are many resources on the web that talk about how to write a scientific discussion. One good one summarizes the idea of a discussion in this way. Explain whether the data support your hypothesis; acknowledge any anomalous data, or deviations from what you expected; derive conclusions, based on your findings, about the process you're studying; relate your findings to previous work in the field (if possible [not so important in a prac report]) and explore the theoretical and/or practical implications of your findings (2).
Questions:
How does the kinetic parameters Km and Vmax obtained from a Lineweaver-Burk plot vary to that of an Eadie-Hofstee and Woolf plot of the same data?
The Lineweaver-Burk plot and the Eadie-Hofstee plot are used in enzyme kinetics to analyze the relationship between substrate concentration and reaction rate. While the Lineweaver-Burk plot shows the reciprocal of the reaction rate against the reciprocal of the substrate concentration, whereas the Eadie-Hofstee plot shows the velocity against the ratio between velocity and substrate concentration and the Woolf plot shows the substrate concentration against the ratio between substrate concentration and velocity. In a Lineweaver-Burk plot Km is represented by the x-intercept whereas Vmax is represented by the y-intercept. In the Eadie-Hofstee the velocity-axis intercept is represented as Vmax whereas in the Woolf plot the intercept formed on the ratio between velocity and substrate concentration is equal to 1/Km.
After studying all graphs, personally the Lineweaver-Burk plot is clearer with representing data however, the Eadie-Hofstee and Woolf plots may be more accurate when it comes to determining Km and Vmax.
There are three types of inhibition of enzyme activity. What are they and how can you determine experimentally what they are? One type of inhibition is theoretical for enzymes that only catalyze a single substrate, which type of inhibitor is this and why is this type of inhibition classified as being theoretical?
There are three types of inhibition which are competitive inhibition, non-competitive inhibition and uncompetitive inhibition.
References
ICMJE international committee of medical journal editors; www.icmje.org
The Writing Centre (website), Scientific Reports, University of North Carolina http://writingcenter.unc.edu/resources/handouts-demos/specific-writing-assignments/scientific-reports
