Enzyme assay analysis of succinate dehydrogenase to resolve Km and Vmax values and to determine the affects of different variables on the oxidation of succinate to fumerate
Introduction
Enzyme kinetics studies chemical reactions where enzymes are involved; rate of reaction and other factors that affect it such as increasing substrate concentration, enzyme concentration, presence of an inhibitor, deviation of temperature and pH. This will highly aid the information about mechanism of an enzyme and its effect on the substrate, subsequently, acquiring a deeper understanding of the system in which enzyme acts upon on; e.g. how metabolism is controlled.
The mitochondria contain enzymes that are involved in cellular respiration where amino acids,
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polysaccharides and fatty acids are processed into ATP, CO2, and water. The tricarbocylic acid (TCA) cycle compromises of a series of reaction heavily involving enzymes within the mitochondria. These are responsible for ATP production during a series of oxidation- reduction reactions beginning with pyruvate and ending with acetyl-coA. The enzyme succinate dehydrogenase(SDH) is accountable for the catalysis of the reduction of its coenzyme, Flavin adenine dinucleotide (FAD) to FADH2 where in return, succinate-succinate dehydrogenate complex is formed and results in the product of fumerate during the dehydration process/ oxidation (Engelkirk et al., 2011). Due to the high importance of SDase, mutation of this would cause problems clinically. For instance, mutation of the gene that encodes SD’s subunit could cause malignant paraganglioma and pheochromocytoma which is a type of a tumor that directly affects the andrenal glands resulting distruption on the control of heart rate, blood pressure and metabolism (Timmers et al., 2007). The aim of this practical was to measure the rate of reaction of succinate dehydrogenase (in mitochondrial suspension) and the effect of variables such as amount of substrates, presence of inhibitor and amount of enzyme are considered.
This was done quantitatively by monitoring the reduction of an artificial electron acceptor, DCIP. As DCIP is initially coloured blue, the reduction gradually turns this to transparent colourless as the reaction continues. Absorption values were recorded at set intervals and used to construct a substrate concentration- velocity graph. This is used to find the Vmax which is the maximum velocity in which the enzymes present can work. The Michaelis constant, Km is the substrate concentration at 1/2Vmax is also calculated from this. Using these value along with the Michaelis Menten model, will aid the understanding of the mechanism by providing a measure of affinity of succinate dehydrogenase (SDH) with its substrate, succinate and subsequently, the efficiency of the reaction.
Results
δAbs at 5 min δAbs at 10 min δAbs at 15 min δAbs at 20 min δAbs at 25 min δAbs at 30 min
Tube A 0.042 0.323 0.592 0.717 0.830 0.892
Tube B 0.114 0.917 0.949 0.955 0.956 0.957
Tube C 0.093 0.543 0.625 0.919 0.947 0.952
Tube D 0.169 0.234 0.410 0.416 0.438 0.445
Tube E 0.044 0.057 0.106 0.115 0.201 0.227
Tube F 0.039 0.044 0.129 0.133 0.134 0.133
Tube G 0.045 0.046 0.064 0.105 0.134 0.134
Table 1 shows that the difference in absorption increases as time progresses.
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Tube B has the largest increase amongst the tubes whilst Tubes E (no Azide), F (no succinate) and G (with boiled mitochondrial suspension) show no significant difference in their recorded absorption throughout the experiment. Abs 0 min Abs 5 min Abs 10 min Abs 15 min Abs 20 min Abs 25 min Abs 30 min V0 at 15 min Tube A 1.115 0.975 0.897 0.869 0.873 0.831 0.724 0.016 Tube B 1.117 0.892 0.613 0.577 0.375 0.273 0.120 0.036 Tube C 1.120 0.635 0.492 0.310 0.289 0.193 0.135 0.054 Tube D 1.125 0.372 0.263 0.165 0.152 0.139 0.127 0.064 Tube E 1.120 0.323 0.287 0.115 0.107 0.101 0.098 0.067 Tube F 1.050 0.223 0.104 0.045 0.037 0.035 0.025 0.067 Table 2 shows that the V0 increases steadily as substrate concentration increases from tube A to tube C whilst the remaining tubes doesn’t display much change in V0. The shape of the graph is very similar to a sigmoidal curve where in the beginning there is a steady, linear increase in velocity where the increase of succinate concentration is proportional to the increase of initial velocity however, at around 0.06 M of succinate, the trend line begins to plateaus and the proportionality between the two variables doesn’t apply anymore hence increasing of succinate concentration does not make a difference in the velocity. The Vmax is 0.067 rpm and Km is 0.019 M. Analysis Table 1 shows that the pattern of tube A,B and C are similar where the difference in absorbance increases over time.
Tube B has the highest increase in absorbance due to the great amount of mitochondrion present whereas tube A and C has less. This would mean that there are more succinate-SDH complexes; in return, more succinate being oxidized to fumerate, thus the reduction of E-FAD to E-FADH2 (DCIP) is greater in tube 2. Malonate is a compound which acts as a competitive inhibitor, it’s chemical structure is similar to succinate, allowing it to bind to the active site of SDH, its presence is seen in tube 4; this restricts the formation of enzyme-substrate complexes being made and consequently, less reaction of succinate to fumerate resulting a significantly less increase of the absorbance recorded over time compared to tubes A,B and
C. During the experiment, sodium azide was used to block the normal electron transport system to monitor reduction; lack of sodium azide would suggest that there are unmonitored fumerate productions explaining the low absorbance seen in tube E as some electrons go through ubiquinone, cytrochrome c- the normal electron path rather than DCIP. There are no significant changes in absorbance in tubes F and G or the absorbance values are too minute to say that reduction of E-FAD is taking place. This is due to absence of substrate in tube F and the result of boiling the mitochondrial solution in tube G. Boiling the mitochondrial solution would have resulted the tertiary structure of the enzyme to denature, enabling it to catalyse the reaction, any change in absorbance from these tubes could be due to the spontaneous breakdown of the substrate or perhaps the DCIP is slightly light sensitive. The Michaelis-Menton equation implies that the velocity of a reaction is directly proportional to the amount of enzyme and substrate through these equations (Pratt and Cornely, 2004): E+S⇄[E+S]→P+E where E= enzyme, S= substrate, P= product and [E+S] is enzyme-substrate complexes. In addition, k-1 and k2 represent [E+S] disssociation and k1 represents [E+S] formation. This equation directly relates to the Km as: Km=(k-1+k2)/k1 Essentially, Km would suggest a measure of affinity and dissociation. A great Km is resulted from a huge number of [E+S] dissociation and a small number of [E+S] formation and vice versa for a poor a Km value. A low Km value would then mean that the affinity of an enzyme for its substrate is high (Traut, 2008). Applying this theory to the results in Figure 1; the Km value for succinate dehydrogenase is 0.019 M, as it is a low number it’s easy to assume that SDH has a great affinity for succinate in the experiment carried out. Analysing shape of Figure 1, the initial linear line given from 0-0.035 M of succinate, would suggest the first order kinetics. Here, there are spare enzymes to be occupied thus increasing the substrate concentration results in more enzymes utilised hence more product and greater velocity; doubling the substrate concentration would result in double the velocity, showing a direct relationship. Where the plateau begins in 0.06-0.1 M would suggest the zero order kinetics being put into place where increasing the substrate concentration won’t have an effect on the velocity any longer, this is due to the enzyme saturation where all the enzymes are already taken and it’s working on its maximum capacity and gives the Vmax of 0.067 rpm (Gaw, 2005). To summarise, SDH works very efficiently with succinate; increasing the substrate would result in more fumerate being produced and increase the velocity of reaction. However, there are restrictions to this such as limiting factors; if the stage where full enzyme saturation is reached or if inhibitor such as malonate is added as this slows down the Vo of reaction due to their interaction with SDH. Reference Engelkirk, P., Duben-Engelkirk, J., and Burton, G. (2011). Burton's microbiology for the health sciences (Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins). P107-113. Gaw, A. (2005). Clinical biochemistry (Edinburgh [u.a.]: Churchill Livingstone). Pratt, C., and Cornely, K. (2004). Essential biochemistry (Hoboken, NJ: J. Wiley). P105-110. Timmers, H., Kozupa, A., Eisenhofer, G., Raygada, M., Adams, K., Solis, D., Lenders, J., and Pacak, K. (2007). Clinical Presentations, Biochemical Phenotypes, and Genotype-Phenotype Correlations in Patients with Succinate Dehydrogenase Subunit B -Associated Pheochromocytomas and Paragangliomas. The Journal Of Clinical Endocrinology & Metabolism 92, P779-786. Traut, T. (2008). Allosteric regulatory enzymes (New York: Springer). Appendix Raw data included in the hard copy.
However, at 3% substrate concentration, the hydrogen peroxide decomposition showed an immediate peak of up to 3.8 mm in height. As the substrate concentration slowly increased, enzyme
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