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BCH2IBM Enzyme Kinetics Assignment
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BCH2IBM Enzyme Kinetics Assignment
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Course Code: BCH2IBM
University: La Trobe University
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Country: Australia
Questions:
1.Describe the aspect of alcohol dehydrogenase behaviour that you plan to investigate in your experiments, and provide the aim/objective of your experimental work.
2. Propose one hypothesis related to the aspect of ADH behaviour that you plan to investigate. A hypothesis is a statement beginning with “That… “, and it must be ‘testable’ by your experiment.
Answer:
Introduction
Enzyme kinetics deals with the study into the chemical reactions which undergo catalysis by enzymes. Enzymes are defined as molecules or proteins that have the ability to change other molecules or reactions (Beneyton et al., 2016). They are catalysts in the sense that they are able to change the other molecules as well as their rate of reactions and they remain unused or unchanged in the process. The molecules that are affected by enzymes are called substrates. Substrates work by binding themselves to the active sites of the enzyme where they are changed into products through various processes and mechanisms known as enzymatic mechanisms. Enzymes perform numerous functions and take part in lots of processes (Obexer et al., 2015). The equation below illustrate the basic mechanism of an enzyme
In which E defines the enzymes, S the substrate and P the product of action of the enzyme, E on the substrate S. from the equation it can be observed that the enzyme binds to the substrate to generate a complex of enzyme substrate which is a revolutionary state where the substrate the changes to be the product that is released (Choi & Chang 2015).
This experiment made use of Michaelis-Menten kinetics in the definition and description of the rate at which a reaction takes place. In most cases, the rates of enzyme reactions never illustrate a linear response if the substrate is increase and hence the process of taking measurements of the initial rate, Vo, over a range of various concentrations of substrates often denoted as [S] is adopted (Damodharan et al., 2015). An increase in the [S] leads to a corresponding increase in the rate of reaction up to a point where the enzyme os saturated with the substrate.
Under such circumstances, the reaction rate plateaus and such s point is known as the Vmax point is defines the maximum rate of the enzyme. The Michaelis-Menten constant, denoted as KM refers to the concentration of the substrate when Vo is equivalent to ½ Vmax. Km is used in the quantification of the ability of an enzyme to catalyze any reaction (Ji et al., 2016). A lower KM value illustrates a better ability of the enzyme in the creation of a product that has small amounts of the substrate.
This experiment gave focus the rate of reaction of the enzyme alcohol dehydrogenase, ADH, that had been isolated from yeast (Matsumura et al., 2015). This enzyme in most cases is used not the catalysis of ethanol and generates acetaldehyde. Reaction involved in the process.
In which ethanol acts as the substrate, Nicotinamide adenine dinucleotide (NAD), serves as the oxidized enzyme. Nicotinamide adenine dinucleotide is the reduced form while acetaldehyde is the product that is generated as a result of the catalysis of the alcohol by the alcohol dehydrogenase enzyme (Vnukov et al., 2015). The absorbance of the product Nicotinamide adenine dinucleotide was taken into consideration in a bid to assay this reaction at a wavelength of 340 nm. Such a wavelength was ideal as it was proportional to the quantity of the Nicotinamide adenine dinucleotide and hence proportional to the quantity of the ethanol that was changed to the final product. The equation below can be used in the description .
With the aid of this assay, an experiment was conducted in which the variables were altered in order to gain an understanding of how enzyme kinetics works. The initial stage involving looking at the how the reaction would proceed in the absence of an enzyme so as to have a basis of performing the experiment (Vnukov et al., 2015). The next stage involved an assessment into the activity of the enzyme using varied concentrations of the substrate.
There were differences in the activity of the enzyme at various concentrations of substrates which illustrated a progress curve that plateaued when the apparent maximum rate, Vmax was reached. The final stage of this experiment involved an experiment that aimed a testing the standard amount of substrate concentration under different temperature values in which the test was done to make a comparison between 4o degrees Celsius and 80 degrees Celsius (Choi & Chang 2015).
Materials and Methods
Week 1 Procedure
This procedure was started by testing a blank solution within the spectrophotometer at a wavelength of 340 nm. The blank was composed of 1.6 ml of water, 0.7 ml of buffer, 0.5 ml of 2M ethanol as well as 0.1 ml of NAD, all of which were placed in a cuvette and passed through the spectrophometer. Such a reaction yielded no results just as was expected and thus served as a control and illustrated that the reaction needed the enzyme.
The next step of this procedure involved the inclusion of the enzyme and using it with various concentrations of the substrate. Each of the concentrations was individually tested in cuvettes using buffer, ADH enzyme and NAD. The first step to generating these cuvettes involved the addition of water, buffer, ethanol and NAD upon which after all was added a time was readied. The coveter was put inside the spectrophotometer and the time started as soon as the ADH was added (Vnukov et al., 2015).
The readings of the absorbance were taken at intervals of 15 seconds at the initial stages for approximately 5 minutes and the reading continued to gradually decrease; alterations were made to take the measurements of the readings at intervals of 30 seconds. This was the used in the determination of the raw initial rates for each and every substrate concentration by measuring the absorbance at 45 seconds and finds the difference of the absorbance at 15 seconds thereafter dividing the result by 30 seconds (Kyznetsova et al., 2015). These were termed as the raw initial rates as they are absorbance but not the concentrations of the products. Beer’s law was used in the conversion of the concentrations of the products and molar extinction coefficient of 6200cm/M was used for NADH.
Beer’s law c (concentration) =Absorbance at 340 nm/ (6200 cm/M). This allowed the conversion of the unit less values of absorbance into molar concentration of the products. A Lineweaver-Burk Plot was set up through computing 1/[S] on the x-axis and computing 1/Vo on the y-axis as soon as the initial rates for each of the substrate concentrations was determined. Vo is derived from the calculations obtained using the Beer’s law and absorbances.
Week 2 Procedure
Week two involved conducting an experiment on the effects of temperature on the activity off an enzyme. The experiment began by the preparation of a standard assay of 1.6 ml of water, 0.7 ml of buffer, 0.5 ml of 2M ethanol as well as 0.1 ml of NAD which was then transferred into 2 cuvettes excluding ADH and NAD. The assay was subjected to varied temperatures of 40?C and 80?C before the enzymes were included. Each of the cuvettes was left in its respective incubation temperature for about 10 minutes before being removed and having the enzyme introduced.
Upon the addition of the enzyme, the cuvette was swiftly placed in the spectrophotometer and the readings of the absorbances taken at intervals of 15 seconds for period not less than 1 minute. The raw initial rates were then determined using the very process as was used before in taking the absorbances at 45 seconds and finding the difference from the absorbance at 15 seconds and dividing the resultant by 30 seconds. The Beer’s law was once more used in the conversion to molar concentrations (Hadwan & Abed 2016). A graph was then plotted of the initials rates against the temperature at which they were incubated.
Results
Lineweaver-Burk Plot
Eadie-Hofstee Plot
Vmax
Km
Vmax
Km
Room Temperature
0.227
8.34
0.238
9.248
40 degrees Celsius
0.204
27.4
0.161
18.2
80 degrees Celsius
-7.78
26.5
0.0716
-50.6
Table 1: Summary table for the values of Vmax and Km derived from Lineweaver-Burk plot and Eadie-Hofstee Plot at three different temperatures
Best Estimate
Vmax
Km
Room Temperature
0.233
9.248
40 degrees Celsius
0.184
18.2
80 degrees Celsius
0.0358
-50.6
Table 2: Table for best estimate of Vmax and Km at three different temperatures
The data in a plotted chart for every assay at different concentrations of the substrates over the time at room temperature. The enzyme was added to the reaction at the origin plot when the time is at 0 seconds. The data points given illustrate those of the absorbance that was picked up at difference rates as the concentration changed over the time (De Rosa et al., 2015). This data is showing progress curved from the ones which were extrapolated raw initial rates.
Figure 3 is Lineweaver-Burk Plot at room temperature. In the figure, the raw initial rates were converted to the actual initial rates using Beer’s law. The law enables changing the unit-less absorbance values into various concentrations making it possible to have the initial rates in concentration values. The Lineweaver-Burk Plot is plotted as 1/initial rate being on the y-axis and 1/substrate concentration on the x-axis.
Figure 8 is the Eadie-Hofstee Plot at room temperature which was derived by plotting the reaction rate as a function of the ratio existing between the substrate concentration and the rate which gave the Vmax at the y-intercept while the x-intercept gives the value of Vmax/Km and Km being the negative slope (Choi & Chang 2015).
The data in figure 6 is a plotted chart for every assay at different concentrations of the substrates over the time at 40?C. The enzyme was added to the reaction at the origin plot when the time is at 0 seconds. The data points given illustrate those of the absorbance that was picked up at difference rates as the concentration changed over the time. This data is showing progress curved from the ones which were extrapolated raw initial rates (Klein et al., 2016).
the raw initial rates were converted to the actual initial rates using Beer’s law. The law enables changing the unit-less absorbance values into various concentrations making it possible to have the initial rates in concentration values. The Lineweaver-Burk Plot is plotted as 1/initial rate being on the y-axis and 1/substrate concentration on the x-axis.
Celsius which was derived by plotting the reaction rate as a function of the ratio existing between the substrate concentration and the rate which gave the Vmax at the y-intercept while the x-intercept gives the value of Vmax/Km and Km being the negative slope.
The data in figure 9 is a plotted chart for every assay at different concentrations of the substrates over the time at 80?C. The enzyme was added to the reaction at the origin plot when the time is at 0 seconds. The data points given illustrate those of the absorbance that was picked up at difference rates as the concentration changed over the time. This data is showing progress curved from the ones which were extrapolated raw initial rates.
the raw initial rates were converted to the actual initial rates using Beer’s law. The law enables changing the unit-less absorbance values into various concentrations making it possible to have the initial rates in concentration values. The Lineweaver-Burk Plot is plotted as 1/initial rate being on the y-axis and 1/substrate concentration on the x-axis.
Figure 8 is the Eadie-Hofstee Plot at 40 degrees Celsius which was derived by plotting the reaction rate as a function of the ratio existing between the substrate concentration and the rate which gave the Vmax at the y-intercept while the x-intercept gives the value of Vmax/Km and Km being the negative slope.
Discussion
The results indicated that the activity of the enzyme is specific to some temperature. The relationship however does not tend to be a linear regression. An increase in the temperature tends to facilitate an increase in the productivity of an enzyme but beyond 45?C the effectiveness drops sharply (Angmo, Kumari & Bhalla 2016). An increase in the temperature leads to a high kinetic energy of the enzyme and the substrate hence the increase in the enzymatic activity. At very high temperatures (80?C), the energy within the enzyme is too high and hence resulting into the denaturing or unfolding of the enzyme. A denatured enzyme is unable to carry out the process of reaction and hence a reduction in the activity of the enzyme. The optimal temperature of the experiment seemed to be around 45?C which was the highest temperature just the enzymes would be denatured.
Conclusion
The temperature experiment illustrated that the ideal temperature for the reaction was about 45?C beyond which the activity of the enzyme would reduce sharply. At optimum temperature, the enzymes have the right kinetic energy for the reaction and hence maximum interaction between the enzymes and the substrate.
References
Angmo, K., Kumari, A., & Bhalla, T. C. (2016). Probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of Ladakh. LWT-food Science and Technology, 66, 428-435
Beneyton, T., Wijaya, I. P. M., Postros, P., Najah, M., Leblond, P., Couvent, A., … & Drevelle, A. (2016). High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics. Scientific reports, 6, 27223
Choi, E. A., & Chang, H. C. (2015). Cholesterol-lowering effects of a putative probiotic strain Lactobacillus plantarum EM isolated from kimchi. LWT-Food Science and Technology, 62(1), 210-217
Damodharan, K., Lee, Y. S., Palaniyandi, S. A., Yang, S. H., & Suh, J. W. (2015). Preliminary probiotic and technological characterization of Pediococcus pentosaceus strain KID7 and in vivo assessment of its cholesterol-lowering activity. Frontiers in microbiology, 6, 768
De Rosa, P., Marini, E. S., Gelmetti, V., & Valente, E. M. (2015). Candidate genes for Parkinson disease: lessons from pathogenesis. Clinica Chimica Acta, 449, 68-76
Fullár, A., Dudás, J., Oláh, L., Hollósi, P., Papp, Z., Sobel, G., … & Kovalszky, I. (2015). Remodeling of extracellular matrix by normal and tumor-associated fibroblasts promotes cervical cancer progression. BMC cancer, 15(1), 256
Hadwan, M. H., & Abed, H. N. (2016). Data supporting the spectrophotometric method for the estimation of catalase activity. Data in brief, 6, 194-199
Ji, Y., Xu, Z., Zou, D., & Gao, K. (2016). Ecophysiological responses of marine macroalgae to climate change factors. Journal of applied phycology, 28(5), 2953-2967
Ji, Y., Xu, Z., Zou, D., & Gao, K. (2016). Ecophysiological responses of marine macroalgae to climate change factors. Journal of applied phycology, 28(5), 2953-2967
Klein, A. M., Mazutis, L., Akartuna, I., Tallapragada, N., Veres, A., Li, V., … & Kirschner, M. W. (2015). Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell, 161(5), 1187-1201
Kyznetsova, M. Y., Makieieva, O. M., Lavrovska, D. O., Tymoshenko, M. O., Sheverova, D. P., Halenova, T. I., … & Ostapchenko, L. I. (2015). Effect of aqueous extract from phaseolus vulgaris pods on lipid peroxidation and antioxidant enzymes activity in the liver and kidney of diabetic rats. Journal of Applied Pharmaceutical Science, 5(5), 001-006
Matsumura, S., Kun, Á., Ryckelynck, M., Coldren, F., Szilágyi, A., Jossinet, F., … & Griffiths, A. D. (2016). Transient compartmentalization of RNA replicators prevents extinction due to parasites. Science, 354(6317), 1293-1296
Obexer, R., Godina, A., Garrabou, X., Mittl, P. R., Baker, D., Griffiths, A. D., & Hilvert, D. (2017). Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase. Nature chemistry, 9(1), 50
Vnukov, V. V., Gutsenko, O. I., Milutina, N. P., Ananyan, A. A., Danilenko, A. O., Panina, S. B., & Kornienko, I. V. (2015). Influence of SkQ1 on expression of Nrf2 transcription factor gene, ARE-controlled genes of antioxidant enzymes and their activity in rat blood leukocytes. Biochemistry (Moscow), 80(5), 586-591
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