Enzymes of the TCA Cycle: Spectroscopic Analysis of Malate and Succinate Dehydrogenase Assay

Enzymes of the TCA cycle 1
ENZYMES OF THE TCA CYCLE
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Enzymes of the TCA cycle 2
Abstract
The Tricarboxylic acid (TCA) cycle is a major metabolic pathway accountable for
providing reduction potential for oxidative phosphorylation and anabolic substrates or cell repair,
growth and proliferation. The following experiment used a spectroscopic method of analysis to
measure the absorbance of malate and succinate dehydrogenase assay. The rate of decrease in
the absorbance was due to NADH oxidation which was the measure of the reaction rates.
Throughout the experiment, the fractions were stored on ice to avoid degradation. It is worth
noting, the rate of decrease or absence of substrates provided the black. Then, after the results of
the absorbance against the concentration over time were plotted, a linear graph with a negative
gradient was obtained.

Enzymes of the TCA cycle 3
Introduction
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

Enzymes of the TCA cycle 4
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.