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Discuss about the Enzymes of the Tea Cycle.

Enzymes of the Krebs cycle and their functions in mitochondrial matrix

The Krebs cycles enzymes are membranes proteins found within the matrix of the mitochondrial except for succinate dehydrogenase which is essential membrane protein locked to the inner mitochondrial membrane (Chandel 2015, pp. 204). Acetyl-CoA joins with oxaloacetate by citrate synthase, to create a 6-C molecule. Therefore, the compound releases citric acid from the enzyme complex.  The fragment of water moves from the third position on the citric acid molecule and add to the fourth position by the enzyme aconitase resulting in isocitrate. Isocitrate dehydrogenase compound catalysis the oxidation of the fourth position of OH group of isocitrate, to produce alpha-ketoglutarate where one NAD molecule changes to NADH. Decarboxylation happens to the alpha-ketoglutarate, changing another molecule of NAD to NADH, by alpha-ketoglutarate dehydrogenase, producing succinyl CoA which is an unstable molecule (Intlekofer et al. 2015, pp. 305). Succinyl-CoA synthesises the addition of a free phosphate group to guanosine diphosphate, generating guanosine triphosphate. Thus, in the course, the CoA group releases from succinyl-CoA, and the resulting molecule is succinate (Shi and Tu 2015, pp.127). The release of two hydrogen atom from succinate occurs when the succinate dehydrogenase reduces FAD to form FADH2, where the yield of the reaction builds fumarate (Ferro, Rodrigues and De Souza 2015, pp. 258).  The final result of the cycle comprises regeneration of oxaloacetate by oxidation of L-malate by malate dehydrogenase where the conversion of one of the molecules of NAD to NADH (West et al. 2015, pp. 553).

For that reason, this report aims to determine dehydrogenase activity utilising artificial oxidate such as dichlorophenolindophenol (DCPIP) for the assay. The paper assess the succinate dehydrogenase distribution between the microsomal (microsomes and cytosol) and mitochondria fractions. Finally, the report illustrate that two forms of malate dehydrogenase are present in yeast cells, one formation predominately in the mitochondria and other in the cytosol. In this experiment, yeast (Saccharomyces cerevisiae) culture is grown, harvested, and disrupted in a French press. Then, fractionation of homogenate into microsomal and mitochondrial will result, whereby the fraction will be subdivided into small aliquots, snap frozen and kept in liquid nitrogen, to avoid rapid degradation.

For both assays, we will utilise the spectroscopic method of analysis at the absorbance wavelength of 340nm and 600 nm for the malate dehydrogenase and succinate dehydrogenase respectively.  For the malate dehydrogenase, one will use 4mg/ml of NADH, 50mM phosphate buffer of pH 7.4 and 1.3mg/ml oxaloacetate.  On the side of the succinate dehydrogenase, 50mM phosphate buffer,  50 ml of 1.5mM DCPIP, 20ml of 12.5mM phenazine methosulphate, 30ml of 20mM KCN, and finally, subcellular fractions of mitochondrial and diluted fraction mitochondrial fractions and microsomal is used.

Sucinate dehydrogenase distribution between mitochondrial and microsomal fractions


Figure 1: Absorbance (340nm) vs. concentration over time (minutes), spectroscopic method of analysis at the absorbance wavelength of 340nm for the malate dehydrogenase, one will use 4mg/ml of NADH, 50mM phosphate buffer of pH 7.4 and 1.3mg/ml oxaloacetate.

Figure 2: Absorbance (600nm) vs. concentration over time (minutes) for the succinate dehydrogenase, 50mM phosphate buffer, 50 ml of 1.5mM DCPIP, 20ml of 12.5mM phenazine methosulphate, 30ml of 20mM KCN

Malate dehydrogenase assay:  the absorbance at 340nm on both mitochondrial fraction and microsomal fraction are high at time zero, before the addition of the substrate. As the time moves, the absorbance of both fractions drops. On the side of the succinate dehydrogenase, the scenario is the same as one for the malate dehydrogenase; the absorbance decreases with the addition of the substrate.

The reduction of oxaloacetate by NADH can be used as the measurement of malate dehydrogenase (Martínez-Reyes et al. 2016, pp. 200).  It is worth mentioning that the reaction of oxalate and NADH in the presence of hydrogen ions lies far to the right.  At a neutral pH and in a slight excess of NADH, the reaction tends to be rapid, and oxaloacetate is quantitatively converted to malate. Therefore, the rate of decrease in absorbance at the 340 can be accounted on the NADH oxidation which entails the measurement of the speed of the reaction.  It is also worth noting that the mitochondrial fraction will comprise some NADH oxidase activity (West et al. 2015). A dilute mitochondrial fraction is used for this essay as the activity of malate dehydrogenase is relatively high. Thus, the NADH oxidase activity under the above conditions is low and does not typically interfere with the assay.

DCPIP and PMS can be reduced under an anaerobic condition to stable forms in the presence of oxygen. PMS acts as intermediary electron carrier where succinate is the substrate. Succinate dehydrogenases catalysis the succinate oxidation to fumarate with the electrons passed to the oxidised DCPIP (Birsoy et al. 2015, pp. 540). The disappearance of oxidised DCPIP was followed spectrophotometrically at 600 nm, which represent the decrease in the absorbance in the above graph. Therefore, the above statement fulfils the objective of an experiment which assesses the succinate dehydrogenase dispersion between the microsomal and mitochondria. 

Conclusion

Yeasts are ubiquitous unicellular fungi widespread in natural environment colonising from terrestrial, aerial to aquatic surrounding, where the active colonisation is intimately connected to their physiological adaptability to an extremely variable atmosphere. In yeast, just like any other heterotrophic organism, the carbon and energy metabolism are inextricably interlinked. ATP is offered by the organic molecules oxidation that also acts as carbon sources for biosynthesis, and ultimately it is used as energy for all sorts of cellular work. All enzymes are localised with one or more particular compartment of the cells. Two significant tactics can be employed to assess the enzymes localization; cell disruption to release the intact organelles, and preservation of the entire cell structure and detection of enzyme activity by histochemical methods coupled with a microscopic assessment.

Two forms of malate dehydrogenase present in yeast cells

In the succinate dehydrogenase, the disappearance of oxidised DCPIP is well depicted spectrophotometrically as the absorbance decreases as the substrate is added.  The function of PMS is to by-pass the cyanide-inhibited site of electron transport, cytochrome oxidase. Therefore, electrons flow from PMS to DCPIP, whereas in vivo electrons flow from FADH to coenzymes Q, cytochrome, cytochrome oxidase and finally to oxygen (Pietrocola, Galluzzi, Bravo-San Pedro, Madeo and Kroemer 2015, pp. 806).  The absorbance of both mitochondrial and microsomal reduces due to a decrease in the rate as result of NADH, which is the measure of the frequency of the reaction.  The effect of the experiment demonstrates the dehydrogenase activity using DCPIP for the essay.  It also examines the distribution of succinate dehydrogenase in microsomal and mitochondrial.   The problem can arise as a result of inaccurate dilution resulting in a concentration of extract being high. Therefore, this will cause an extremely high malate dehydrogenase.  Additionally, the fractions of the mitochondrial and microsomal are very sensitive to degradation and thus exposing them to high temperature will alter the samples. Thus, the samples should always be stored or kept on ice at all times. 

References

Birsoy, K., Wang, T., Chen, W.W., Freinkman, E., Abu-Remaileh, M. and Sabatini, D.M., 2015. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell, 162(3), Elsevier, pp.540-551. [Online]. Available from:  https://doi.org/10.1016/j.cell.2015.07.016, [Accessed on 17 September 2018].

Chandel, N.S., 2015. Evolution of mitochondria as signaling organelles. Cell metabolism, Elsevier 22(2), pp.204-206. [Online]. Available from:  https://doi.org/10.1016/j.cmet.2015.05.013, [Accessed on 17 September 2018].

Ferro, M.S., Rodrigues, G.M. and De Souza, R.R., 2015. The role of mitochondria in physical activity and its adaptation on aging. Journal of Morphological Sciences, 32(4), pp.257-263. [Online]. Available from:  https://jms.org.br/PDF/v32n4a07.pdf, [Accessed on 17 September 2018].

Intlekofer, A.M., Dematteo, R.G., Venneti, S., Finley, L.W., Lu, C., Judkins, A.R., Rustenburg, A.S., Grinaway, P.B., Chodera, J.D., Cross, J.R. and Thompson, C.B., 2015. Hypoxia induces production of L-2-hydroxyglutarate. Cell metabolism, 22(2), Elsevier,  pp.304-311. [Online]. Available from:  https://doi.org/10.1016/j.cmet.2015.06.023, [Accessed on 17 September 2018].

Martínez-Reyes, I., Diebold, L.P., Kong, H., Schieber, M., Huang, H., Hensley, C.T., Mehta, M.M., Wang, T., Santos, J.H., Woychik, R. and Dufour, E., 2016. TCA cycle and mitochondrial membrane potential are necessary for diverse biological functions. Molecular cell, 61(2), Elsevier,  pp.199-209. [Online]. Available from:  https://doi.org/10.1016/j.molcel.2015.12.002, [Accessed on 17 September 2018].

Pietrocola, F., Galluzzi, L., Bravo-San Pedro, J.M., Madeo, F. and Kroemer, G., 2015. Acetyl coenzyme A: a central metabolite and second messenger. Cell metabolism, 21(6), Elsevier, pp.805-821. [Online]. Available from:  https://doi.org/10.1016/j.cmet.2015.05.014, [Accessed on 17 September 2018].f

Shi, L. and Tu, B.P., 2015. Acetyl-CoA and the regulation of metabolism: mechanisms and consequences. Current opinion in cell biology, 33, Elsevier, pp.125-131, Elsevier, [Online]. Available from:  https://doi.org/10.1016/j.ceb.2015.02.003, [Accessed on 17 September 2018].

West, A.P., Khoury-Hanold, W., Staron, M., Tal, M.C., Pineda, C.M., Lang, S.M., Bestwick, M., Duguay, B.A., Raimundo, N., MacDuff, D.A. and Kaech, S.M., 2015. Mitochondrial DNA stress primes the antiviral innate immune response. Nature, 520(7548), p.553. [Online]. Available from:  https://doi.org/10.1038/nature14156, [Accessed on 17 September 2018].

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