In part I, we decoded half of the Krebs cycle as citrate ion (6C) is converted to succinyl-CoA (4C) after several steps. Afterwards, in succinyl coenzyme synthetase (CSC) succinate (4C) is formed from succinyl-CoA (4C) with helps of either GDP or ADP.
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| Succinyl-coenzyme synthetase mechanism of succinyl-CoA transformation into succinate |
The first step of this transformation is the phosphorylation of succinyl-CoA where CoA-SH is released. During this step, phosphate ion acts as nucleophile attacking the carbonyl thioester giving succinyl-phosphate ester. It is quite interesting in this step as the a thioester which can be easily interconverted to various carboxylic acid derivates. The the phosphate ester is attacked by nucleophilic His-246 residue releasing succinate with one of its oxygen atoms is from phosphate ion. Consequently, the phosphate ion is transferred to His-246 residue which then attacked by NDP (ADP or GDP) producing NTP; in certain point of view CSC can be seen as NTP synthetase. Furthermore, the active site can be used again for another cycle.
The formed succinate (4C) is oxidised (dehydrogenised) forming fumarate (4C) and this process is catalysed by an enzyme complex of succinate dehydrogenase or succinate-CoQ reductase (SQR). The transformation of succinate to fumarate in this enzyme complex happens via redox couple where the transformation itself is oxidation and at the other ubiquinone (coenzyme Q) is reduced to ubiquinol. The enzyme complex SQR consists of four sub-units SdhA, SdhB, SdhC, and SdhD; SdhC and SdhD are transmembrane sub units and they are embedded within the inner membrane of mitochondrion.
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| Redox couple in the transformation of succinate into fumarate in SQR enzyme complex |
The formation fumarate from succinate can be seen as elimination of two hydrogen atoms, one of them as the leaving group. The nature of this elimination is not well-understood but at this moment two pathways are proposed: E2 or E1cb elimination.
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| Two proposed mechanisms in succinate oxidation in SQR |
In both proposed elimination mechanisms, succinate is anchored at the active site by three amino acids (Thr, Arg, and His) while the basic residue is not known. Several candidates for this basic residue have been proposed such as Glu-255, Arg-286, and His-242. In E2 process, the elimination happens in concerted fashion, both deprotonation and reduction of FADH
2 happens at the same time. Meanwhile in E1cb, the deprotonation occurs first forming enolate intermediate followed by the elimination of H
-, i.e. reduction of FAD.
Following the succinate oxidation, the electrons produced are transferred to reduced CoQ and the electron transfer from SdhA to SdhC/SdhD happens via electron tunnelling process. The electrons move along Fe-S clusters, [2Fe-2S] then [4Fe-4S], until reaching [3Fe-4S] cluster where it is then transferred to the active site for CoQ reduction. Then, the two electrons with two H
+, supplied by water molecules, are used to reduce CoQ and giving ubiquinol. It is noteworthy that there is a haem prosthetic group within this enzyme complex but its function is not well-known. One of the proposed functions is the first electron can from [3Fe-4S] to ubiquinone active site may tunnel back and forth between haem group and ubiquinone intermediate. In this way, haem group acts as an electron which prevents the interaction with molecular oxygen to produce reactive oxygen species. Furthermore, it has also been proposed there is gating mechanism that might prevent the electrons tunnel directly from [3Fe-4S] to the haem group. The proposed mechanism mentions His-207 might act as a modulator that control the electron flows from both redox centres.
Then, fumarate (4C) undergoes hydration reaction forming
L-malate (4C)and this transformation is catalysed by fumarase. This transformation might look much simpler then the previous reactions but the enzyme mechanisms of fumarase is not well-understood.
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| The proposed mechanism of fumarate to L-malate transformation catalysed by fumarase |
The proposed mechanism show that isomerisation of the enzyme happens during the process and this is due to the proton transfer during the transformation. The reaction begins when fumarate undergoes conjugate addition by water and this reaction proceeds via general-base catalysis giving enolate intermediate. Besides the enolate intermediate, the first isomerisation of the enzyme occurs. Then, the enolate is protonated by the acidic residue giving
L-malate which then released from the active site. The enzyme isomerise once more during the
L-malate formation and it isomerise once more after proton transfer to repeat the enzymatic cycle.
The next reaction in this cycle is the oxidation
L-malate (4C) by NAD
+ to give oxaloacetate (4C) in enzyme malate dehydrogenase (MDH).
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| The mechanism of L-malate oxidation into oxaloacetate catalysed by malate dehydrogenase |
During this reaction, MDH undergoes conformational change that encloses the substrate to minimise solvent exposure and bringing the substrate to close proximity. The oxidation process is catalysed by the helps of three residues: Asp-168 and His-195 as proton transfer system and arginines (Arg-102, Arg-109, and Arg-171) anchor the substrate in its place.
L-malate is oxidised via deprotonation by His-195 and Asp-168 proton shuttle and then H
- is transferred to NAD
+ forming oxaloacetate and NADH. The hydrogen-bonding network not only securing the substrate in its position but also stabilising the transition state of this reaction which is one of the ways catalysing reaction. Interestingly, this oxidation is a reversible reaction with positive ΔG; thus the concentration of oxaloacetate is relatively lower to
L-malate.
The final reaction in this series is the condensation reaction between oxaloacetate (4C) and acetyl-CoA (2C) regenerating citrate ion (6C). This reaction is catalysed by citrate synthase which exists in nearly all living cells. In short, the citrate synthesis in this cycle proceeds via Claisen condensation reaction.
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| The mechanism of acetyl-CoA and oxaloacetate condensation forming citrate catalysed by citrate synthase |
In citrate synthesis, acetyl-CoA is deprotonated by Asp-375 residue forming an enol of acetyl-CoA which will act as the nucleophile. It is interesting to see acetyl-CoA being deprotonated rather than the oxaloacetate and this might be due to the fact oxaloacetate has -2 charge and the deprotonation process is the removal of positive species, i.e. proton. Then, the enol attacks the oxaloacetate giving citryl-CoA which then hydrolyse to yield citrate. Thus, the cycle can be repeated.
As we've seen in these two parts, the whole Krebs cycle can be understood using our knowledge of mechanisms in organic chemistry, from alkenes chemistry, decarboxylation and carbonyl chemistry which feature heavily in this cycle. Besides the knowledge in organic chemistry, redox couples are also featured in this cycle and the way the cell regulates these reactions ensure living things can survive in this harsh world.
References
- G. Wiegand and S. J. Remington, Annu. Rev. Biophys. Biophys. Chem., 1986 15, 97-117.
- C. R. Goward and D. J. Nicholss, Protein Sci., 1994, 3, 1883-1888.
- V. Yankovskaya, R. Horsefield, S. Törnroth, C. Luna-Chavez, H. Miyoshi, C. Léger, B. Byrne, G. Cecchini, and S. Iwata, Science, 2003, 299, 700-704.
- Q. M. Tran, R. A. Rothery, E. Maklashina, G. Cecchini, and J. H. Weiner, J. Biol. Chem., 2006, 281, 32310-32317.
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