Mitochondrion and TCA cycle

The mitochondrion is usually known to be the powerhouse of any living cells in the body of an organism. It is composed of all the required machinery in the provision of the cells as well as their components with the energy that is needed to carry out the various cellular processes including movement, growth and development among other vital processes of the cell. The matrix of the mitochondrion forms the base on which TCA cycles takes place, where pyruvate  which is oxidize from glucose in glycolysis processes ins changed into acetyl-CoA, which is then fed into the pathway to undergo oxidation releasing carbon dioxide and the conserved (Aspuria et al., 2014).

Succinate dehydrogenase remains the only enzyme rom the TCA cycle which is as well part of the electron transport system hence it is often located on the inner membrane. Succinate dehydrogenase has a co-enzyme, flavin adenine dinucleotiede all of which are represented as a complex E-FAD and work by oxidizing the metabolite succinate to form fumarate. Succinate dehydrogenase eliminates the electrons from succinate leading to a reduction in FAD and thus a decrease in the enzyme complex E-FADH₂. The reduced enzyme in turn transfers electrons to coenzyme Q from which it is takes through the remaining chain of electron transport (Calió et al., 2017).

Succinate dehydrogenase is an enzyme that is located in the inner membrane. The location makes it a very easy target for isolation when conducting a study on the citric acid cycle. The main role of the enzyme in the cell is to oxidize succinate to form fumarate which is then usable as a marker during the process of isolation of mitochondria via differential centrifugation. The isolated mitochondria can then be treated using a sodium azide reagent to prevent transportation of electron of the mitochondrion in the cell extract.

An artificial electron acceptor is often used in taking the measurement of the activity of an enzyme (2, 6-dichlorophenolindphenol, DCPIP), which is used in the acceptance of two electrons. Upon reception of electrons, the oxidised 2, 6-dichlorophenolindphenol undergoes reduction and the colour of the mixture turns to colourless from blue (Dudek et al., 2015). The change in the colour can be quantified suing spectrometry at a range of 600nm which then provides the contents of mitochondria in the given sample.

Enzymes serve as regulators for the various metabolic pathways which reduce the activation energy so as to catalyze acceleration in the rate of biochemical reactions. Most of the enzymes are characterized as demonstrating Michaelis-Menten (M-M) kinetic characteristics. Enzymes work by creating a binding with their substrates in a reversible manner thereby altering the conformation of the substrate leading to the formation of a complex of enzyme-substrate and hence detaching resulting in free enzyme and the product. In cases where the concentration of the substrate is low, little enzyme activity is noticed and thus a slow rate of reaction (Guzzo et al., 2014).

Structure and role of Succinate Dehydrogenase Enzyme

On the contrary, a high concentration of the substrate or when the substrate has a saturated concentration, the enzymes tend to be more active and hence a faster rate of reaction. At some point when the substrate concentration is highly saturated, where is recorded no increase in the rate of the reaction. Alongside the concentration of the substrate, these changes could be featured as the Michaelis-Menten (M-M) constant as well as the maximum velocity (Lampropoulou et al., 2016). These factors form an integral part of the factors which influence the initial velocity of any biochemical reactions and thus contributing to an elaborate understanding of the Michaelis-Menten (M-M) equation.

Nonetheless, in the presence of a competitive inhibitor, the inhibitor is able to bind to the active site as to stop the normal substrate from making a bind and forming the product. This leads to a competition between the substrate and the product for the active site of the enzyme, a dynamic that is based on the Michaelis-Menten (M-M) equation and permits the maximum velocity to remain constant even as the Michaelis-Menten (M-M) constant changes (Kim et al., 2015).

The role of this laboratory experiment was measurement of the activity rate of succinate dehydrogenase in the catalysis of the reaction succinate to form fumarate in vitiro with the aid of mitochondrial fraction extracted from cauliflower cells. The measurement of the rate of the reaction is done through making observations on the reduction of 2, 6-dichlorophenolindphenol which is an artificial electron acceptor as opposed to coenzyme Q.

Sodium azide blocks are added to the electron systems to ensure that the electrons are empowered to reduce the concentration of coenzyme Q (Kitazawa et al., 2017). The electrons are rather transported to 2, 6-dichlorophenolindphenol from E-FADH₂. A colour change of the 2, 6-dichlorophenolindphenol would be used to identify its reduction where the oxidized form of the acceptor is often blue in colour which changes to colourless upon being reduced. The equation of the reduction is as shown below:

E-FADH₂+DCPIP_ox (blue) E-FADH₂+DCPIP_re (colourless)

The degree of oxidation of the 2, 6-dichlorophenolindphenol DCIP is measured using the various absorbances of the various concentrations of enzymes through the use of a spectrophotometer at 600nm.

The tubes 1 to 3 are used in testing the initial velocities of the reaction when it begins from succinate to fumarate of the various concentrations of enzymes having the same quantity of succinate. The initial velocities of the reaction change with changes in the concentration of the enzymes. The tube labelled 4 is used in testing the effect of a competitive inhibitor (Malonate for this experiment) on the level of activity of an enzyme.

Enzymes and Michaelis-Menten Kinetics

The tube labelled 5 does not have sodium azide blocks added to the normal electron path in the chain of electron transport and is thus used for the purposes of making comparison of the rates of reaction against those tubes which contain sodium azide blocks (Lampropoulou et al., 2016). The tube labelled 6  is used in the testing of the rate of reaction in which succinate is not added while the tube labelled 7 has been heated inside a hot water bath so as to kill or denature the enzyme succinate dehydrogenase thereby bringing it to an inactivated state.

This tube is then adopted as the 0-minute mark of reading for all the other tubes in taking the measurement of the changes in the absorbance since as it undergoes cooling; it changes into its original conformation. This serves to be representing the enzymes before the occurrence of a reaction.

The variation in the absorbance across the 35 minutes of the experiments should demonstrate the variations in the rates of reaction as per the differences in the concentration of enzyme. It should as well indicate the rate of a reaction is slowed with the presence of competitive inhibitors or the absences of the enzyme blockers (sodium azide for this experiment) and substrate (Lampropoulou et al., 2016).

The experiment aims at separating the components of the liver cells based on the using differential centrifugation

• Determine which components of the liver cells contain succinate dehydrogenase
• Identification of the factions that contain mitochondria

1. Mitochondria was isolated
2. The suspension was filtered using cheesecloth and the centrifuged at about 600g for 10 minutes at 4?C
3. The post mitochondrial supernatant fluid was discarded
4. The pellet was scraped from the use and the sediment resuspended in the medium of assay using a pipette. It was kept in ice bath
5. The 10 cuvettes were labelled as per the table below. 0.6 mL of the solution was heated for the case of tube 7 for about 5 minutes after which it was cooled in an ice bath.
6. The spectrophotometer was set at 600 nm (Li et al., 2016)
7. The appropriate volume of the medium of assay was added and the different solutions as indicated in the table below were as well added expect mitochondrial suspension.
8. The tube was covered using parafilm and the inverted so as to ensure thorough mixing of the contents
9. The accurate volume of the mitochondrial suspension was then added to the fist cuvette as per the table and the turned upside down so as to ensure thorough mixing.
10. Mitochondrial suspension was added to the subsequent cuvettes after every half a minute. The suspension was not added to the blanks (Li et al., 2016)
11. The spectrophotomer was adjusted using blanks #1 and the absorbance readings measurements taken for tube 1
12. An adjustment was again made this time using blank #2 and the measuring of the absorbance reading for tube 2 taken followed by blank #3 for the tubes labelled 3 to 7
13. The absorbance of all he seven tubes were taken after at interval of every 5 minutes for a period of 5 minutes, ensuring adjustments to the spectrophotometer using ach of the blanks as per the corresponding tubes (Li et al., 2016).

Tube #	5 min	10 min	15 min	20 min	25 min	30 min	35 min
1	0.62	0.46	0.30	0.38	0.25	0.26	0.22
2	0.41	0.25	0.11	0.04	0.03	0.02	0.02
3	0.48	0.36	0.25	0.15	0.07	0.05	0.04
4	0.58	0.55	0.56	0.55	0.54	0.55	0.52
5	0.60	0.54	0.52	0.49	0.47	0.47	0.44
6	0.67	0.75	0.67	0.67	0.83	0.68	0.70
7	0.75	0.79	0.72	0.72	0.73	0.71	0.73

Table 1: Readings of Absorbance at Every Time Interval

The measurement of the absorbance at every interval of 5 minutes is recorded in table 1 for a period of 35 minutes. As can be observed from the table, the tubes 1 to have illustrated an almost the same sequence or patterning of beginning with a relatively high absorbance measurement or reading with decreased at every subsequent time interval. The tubes 6 and 7 demonstrate an irregular pattern and do not exhibit a consistent decrease or increase all over the time frame of the experiment.

Tube #	5 min	10 min	15 min	20 min	25 min	30 min	35 min
1	0.12	0.28	0.44	0.36	0.49	0.48	0.52
2	0.33	0.49	0.63	0.70	0.71	0.72	0.72
3	0.26	0.38	0.49	0.59	0.67	0.69	0.70
4	0.16	0.19	0.18	0.19	0.20	0.19	0.22
5	0.14	0.20	0.22	0.27	0.27	0.27	0.30
6	0.07	-0.01	0.07	0.07	-0.09	0.06	0.04
7	0.00	-0.05	0.02	0.02	0.01	0.03	0.01

Table 2: Overall Change in the Absorbance at Every Time Interval

The changes in the absorbance at each interval of 5 minutes are illustrated in table 2. The date for each time interval for each of the tubes was gathered through finding the difference of the reading of each of the tubes and at the given time and the reading of the absorbance from the interval of 5 minutes for tube 7 (Yu et al., 2017). The tubes 1 to 5 with the exception of tube 4 had a generation trend of beginning where they began n with a relatively low variation at the initial time of 5 minutes and the changes increases which finally levelized off and plateau. There tends to be fluctuations in tube 4, ranging between 0.19 and 0.22. The tubes 6 and 7 just as was the vase with table 1 display no consistency in their changes in the patterns.

Laboratory experiment and method

Figure 1: Total Change in the Absorbance at every Time Interval for tubes 1-4

The changes in the readings of the absorbance at each time interval of 5 minutes for the tubes 1 to 4 are illustrated in figure 1

 

Figure 2: The initial velocity of Concentration of Enzyme for the tubes 1-3

The tubes labelled 1 to 3 indicate a trend of increase for some time and then finally levelling off while tube 4 remains fairly constant for the full time of the experiment. This figure shows as there is an increase in the initial velocity with an increase in the concentration of the enzyme in every tube (Yu et al., 2017).

The initial velocity is directly proportional to the concentration of an enzyme as per Michaelis-Manton equation. This means as the concentration of the enzyme increase, binding of more substrate occurs at a faster rate owing to the high affinity of the enzyme for the substrate, leading to the high initial velocity of the reaction as has been recorded (Mills et al., 2016). This can be observed in the data as illustrated in figure 2 illustrate that the initial velocity of the reaction that leads to the conversion of succinate to fumarate  through the use of succinate dehydrogenase increases with an increase in the concentration of the enzyme. The lowest quantity of suspension of mitochondrion is observed in tube 1 which contains the lowest amount of succinate dehydrogenase (Na et al., 2014). This is the reason why the lowest initial velocity is recorded in tube 1. On the contrary, the tube labelled 2 was observed to be having the highest of all the initial velocities since it was composed of the greatest concentration of the enzymes in the 0.9 mL of suspension of mitochondria.

The impacts of Malonate which was used as a competitive inhibitor was tested by adding it to the tube labelled 4. The tube 3 and 4 had similar concentrations of the enzyme even though tube 4 recorded a relatively slower reaction rate as compared to the tube labelled 3. This is attributed to the effects of Malonate on the enzyme. The molecular structure of the competitive enzyme is similar to that of succinate and hence able to easily bind to the active site of the enzyme succinate dehydrogenase. This leads to prevention of the enzyme from binding and hence the recorded slower initial velocities as well as the rate of reaction. It would take relatively longer time for a successful binding of the succinate to its enzyme (Rizza et al., 2016).

Results and Analysis

The reaction rate from the succinate to the final product fumarate through succinate dehydrogenase is described by the changes in the absorbances as recorded in table 2. The increase in the changes illustrates oxidation of more succinate to form fumarate as well as a reduction of E-FAD to E-FADH₂. The colour changes of the DCPIP solution for blue to colourless as well as the variations in the absorbances illustrated oxidation of E-FADH₂ to reduce DCPIP. Almost at the edn of the full time of the experiment, 35 minutes, the changes begin levelling off and plateau as observed in the figure 1 above (Williamson et al., 2015).

Nonetheless, succinate was not added to the tube labelled 6 and thus the recorded slower rate of reaction as there was limited amount of succulent available in the mitochondria in comparison to the other samples used in the experiment. There was no addition of sodium azide block in tube 5 the stop the electron transport system to check on the reduction of the amount of DCPIP. The changes on the absorbance demonstrated the reduction in the DCPIP where the variation were noticed to be less significant in tube 5 in comparison to those tubes that had sodium azide added as those tubes experienced more transfer of E-FADH₂ to coenzyme Q as compared to DCPIP (Xiao et al., 2016). The suspension of mitochondria in tube 7 underwent heating for about 5 minutes so as to kill the succinate dehydrogenase enzyme and reduce it to its inactive state. As it underwent cooling, it changes to its original conformation which represented the state of an enzyme just before any reaction occurred to change succinate to fumarate.

Conclusion

Following the data gathered in this experiment, a conclusion can be made that an increase in the concentration of succinate dehydrogenase leads to a corresponding increase in the rate of oxidation of succinate leading to the formation of fumarate. Nonetheless, an addition of Malonate as well as lowering the quantity of sodium azide or succinate lead to a reduction in the rate of the reaction since their interactions with the enzyme inhibits binding the enzyme substrate binding.

References

Aspuria, P.J.P., Lunt, S.Y., Väremo, L., Vergnes, L., Gozo, M., Beach, J.A., Salumbides, B., Reue, K., Wiedemeyer, W.R., Nielsen, J. and Karlan, B.Y., 2014. Succinate dehydrogenase inhibition leads to epithelial-mesenchymal transition and reprogrammed carbon metabolism. Cancer & metabolism, 2(1), p.21

Calió, A., Grignon, D.J., Stohr, B.A., Williamson, S.R., Eble, J.N. and Cheng, L., 2017. Renal cell carcinoma with TFE3 translocation and succinate dehydrogenase B mutation. Modern Pathology, 30(3), p.407

Dudek, J., Cheng, I.F., Chowdhury, A., Wozny, K., Balleininger, M., Reinhold, R., Grunau, S., Callegari, S., Toischer, K., Wanders, R.J. and Hasenfuß, G., 2015. Cardiac?specific succinate dehydrogenase deficiency in Barth syndrome. EMBO molecular medicine, p.e201505644

Guzzo, G., Sciacovelli, M., Bernardi, P. and Rasola, A., 2014. Inhibition of succinate dehydrogenase by the mitochondrial chaperone TRAP1 has anti-oxidant and anti-apoptotic effects on tumor cells. Oncotarget, 5(23), p.11897

Kim, E., Rath, E.M., Tsang, V.H.M., Duff, A.P., Robinson, B.G., Church, W.B., Benn, D.E., Dwight, T. and Clifton-Bligh, R.J., 2015. Structural and functional consequences of succinate dehydrogenase subunit B mutations. Endocrine-related cancer, 22(3), pp.387-397

Kitazawa, S., Ebara, S., Ando, A., Baba, Y., Satomi, Y., Soga, T. and Hara, T., 2017. Succinate dehydrogenase B-deficient cancer cells are highly sensitive to bromodomain and extra-terminal inhibitors. Oncotarget, 8(17), p.28922

Lampropoulou, V., Sergushichev, A., Bambouskova, M., Nair, S., Vincent, E.E., Loginicheva, E., Cervantes-Barragan, L., Ma, X., Huang, S.C.C., Griss, T. and Weinheimer, C.J., 2016. Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation. Cell metabolism, 24(1), pp.158-166

Li, Y.H., Choi, D.H., Lee, E.H., Seo, S.R., Lee, S. and Cho, E.H., 2016. Sirtuin 3 (SIRT3) Regulates α-Smooth Muscle Actin (α-SMA) Production through the Succinate Dehydrogenase-G Protein-coupled Receptor 91 (GPR91) Pathway in Hepatic Stellate Cells. Journal of Biological Chemistry, 291(19), pp.10277-10292

Mills, E.L., Kelly, B., Logan, A., Costa, A.S., Varma, M., Bryant, C.E., Tourlomousis, P., Däbritz, J.H.M., Gottlieb, E., Latorre, I. and Corr, S.C., 2016. Succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages. Cell, 167(2), pp.457-470

Na, U., Yu, W., Cox, J., Bricker, D.K., Brockmann, K., Rutter, J., Thummel, C.S. and Winge, D.R., 2014. The LYR factors SDHAF1 and SDHAF3 mediate maturation of the iron-sulfur subunit of succinate dehydrogenase. Cell metabolism, 20(2), pp.253-266

Rizza, S., Montagna, C., Cardaci, S., Maiani, E., Di Giacomo, G., Sanchez-Quiles, V., Blagoev, B., Rasola, A., De Zio, D., Stamler, J.S. and Cecconi, F., 2016. S-nitrosylation of the mitochondrial chaperone TRAP1 sensitizes hepatocellular carcinoma cells to inhibitors of succinate dehydrogenase. Cancer research

Williamson, S.R., Eble, J.N., Amin, M.B., Gupta, N.S., Smith, S.C., Sholl, L.M., Montironi, R., Hirsch, M.S. and Hornick, J.L., 2015. Succinate dehydrogenase-deficient renal cell carcinoma: detailed characterization of 11 tumors defining a unique subtype of renal cell carcinoma. Modern pathology, 28(1), p.80

Xiao, W., Sarsour, E.H., Wagner, B.A., Doskey, C.M., Buettner, G.R., Domann, F.E. and Goswami, P.C., 2016. Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes. Archives of toxicology, 90(2), pp.319-332

Yu, W., Ni, Y., Saji, M., Ringel, M.D., Jaini, R. and Eng, C., 2017. Cowden syndrome-associated germline succinate dehydrogenase complex subunit D (SDHD) variants cause PTEN-mediated down-regulation of autophagy in thyroid cancer cells. Human molecular genetics, 26(7), pp.1365-137