Acetyl-CoA productionAcetyl Coenzyme A is the major common metabolic product of several pathways, including the glycolytic pathway, the metabolism of fatty acids, and the cellular digestion of amino acids. Acetyl-CoA is basically a modified ADP molecule, having the external hydroxyl group substituted with a pantothenic acid residue, also connected to a β-mercaptoethylamine group. In the acetylated form CoA-SH is converted to a thioester, thus opening its possibility of being further oxidised in the cell.
In order to gain access to the TCA cycle, pyruvate undergoes conversion to Acetyl-CoA, a reaction that occurs when catalysed by Pyruvate dehydrogenase multienzyme complex (PDC). PDC acts as a sequence of enzymes whose action is corroborated altogether. It basically involves the reaction of pyruvate and CoA-SH to form Ac-CoA and CO2. By radioactive labelling of CO2 it has been shown that the reaction is irreversible as the labelled CO2 would not reconvert back to radioactively labelled pyruvate.
The PDC multi-enzyme presents a number of advantages when it comes to catalytic efficiency. First and foremost, as the reaction rates are generally limited by the frequency of enzyme-substrate collisions, the PDC has created the circumstances of reducing the distance between the substrate and the enzyme. In this way, once the substrate enters the multi-enzyme complex, it is very easy for it to be passed from one enzyme to another, thus enhancing the reaction rates.
The mechanism of the reaction involves three enzymes working together as integrant partd of the PD complex and five co-factors, four of which happen to be derived from vitamins. These are thiamine pyrophosphate (TPP), flavin adenin dinucleotide (FAD), CoA-SH, NAD and lipoate. The reaction by itself involvs three major steps and two regeneration steps.
The first step of the PDC reaction is the reduction of pyruvate to Hydroxyethyl-TPP•E1. In this step, pyruvate dehydrogenase(E1), a TPP demanding enzyme, decarboxylates the pyruvate into the formation of hydroxyethyl-TPP•E1. However, TPP•E1 does not release acetaldehyde. Instead, the hydroxyethyl-TPP•E1 is sent to the next enzyme in the complex, dihydrolipoyl tranacetylase (E2). The lipoamide group binded through an amide bridge to the lys residue of E2 reacts with the hydroxyethyl-TPP•E1. The outcome of the reaction is the regeneration of TPP and the conversion of the reactant into acetyl-dihydrolipoamide-E2, through an oxidation process.
The third reaction of the PDC series is the last major step of the reaction. The acetyl-dihydrolipoamide-E2 recently created is transesterificated. In thir reaction, the sulfhydryl group of CoA attacks the acetyl group of acetyl-dihydrolipoamide-E2 to form an intermediate that undergoes decomposition to Acetyl-CoA and dihydrolipoamide-E2.
The last two steps of the PDC reaction are regeneration steps in order to reconvert dihydrolipoamide-E2 to lipoamide-E2. With this view, dihydrolipoamide-E2 is first oxidised in a reaction at the expense of FAD, regenerated itself by its reaction with NAD+.
Experimental Evidence for the existence of the TCA CycleSince the elucidation of glycolysis in the early ‘30s, not much progress had been made in the field of glucose oxidation, especially while considering its conversion to carbon dioxide and oxygen uptake. However, what was already known was that several metabolic intermediates play a certain role in this process but it wasn’t still clear how do these reaction take place and how are they interconnected in the metabolic pathway that deals with glucose digestion.
Then, in 1935, the Hungarian researcher Albert Szent-Gyorgyi proved experimentally that small amounts of succinate, fumarate, L-malate, or oxaloacetate would determine a drastic increase of cellular respiration and oxygen uptake in finely cut muscle cells. He suggested that the compounds were converted from one to another according to the sequence: Succinate → fumarate → L-malate → oxaloacetate. In the same year, C. Mauritius and F. Knoop discovered another part of the tricarboxylic acid cycle, from citrate → cis-aconitate → isocitrate → α-ketoglutarate.
Since it was already known that α-ketoglutarate can in certain catalytic conditions be converted into succinate with the release of a molecule of CO2, what was necessary now, was to close the circle, to bring sufficient proof that oxaloacetate would undergo a certain reaction in order to convert itself into citrate. As Mauritius and Knoop demonstrated that the reaction is theoretically possible in non-enzymatic conditions (H2O2 in basic environment), Krebs formulated a hypothesis that these reactions would form a cycle. He sustained the hypothesis by arguing that the reduction of fumarate to succinate is inhibited by malonate, therefore a cyclic pathway could exist in order for this conversion to become possible, as predicted by his pigeon breast muscle respiration experiments. Moreover he inferred that this would also represent the main pathway for pyruvate oxidation in muscle given the reaction rates observed.
The Cycle and its stepsOnce Acetyl-CoA has been produced, the tricarboxylic acid cycle can take place. As we are discussing the steps of a cycle, in the first step of the cycle, Acetyl-CoA will suffer condensation with the oxaloacetate produced in the last step of the cycle. The product of this initial reaction is citrate, also giving the name of this cyclic metabolic pathway.
The reaction is catalysed by citrate synthase, also known as citrate condensing enzyme. It is a dimer enzyme that binds Acetyl-CoA and oxaloacetate respectively in two steps. In the first step, oxaloacetate is binded to this enzyme in the deep cleft to be found at the junction between the two subunits that form the enzyme. Therefore, the enzyme is transformed through a conformational change from the open state in which it stood until right before the binding of oxaloacetate, to a closed state, moment in when Acetyl-CoA can also bind to the synthase. The order of the binding in this enzyme has been experimentally proven by the competitive inhibition of the enzyme by Acetonyl Co-A (CoAS-CH2C(O)CH3) or by carboxymethyl-CoA (CoAS-CH2COOH). By binding these two components, the enzyme catalyses an aldol Claisen condensation. Within the mechanism, the Acetyl- CoA enol formed is stabilised by a low-barrier hydrogen bond formed with the Hydrogen atom the Histidine residue found in the active site of the protein.
The last step of the cycle involves a reversible reaction whose equilibrium is displaced far to the left. Luckily, the reaction rate is sufficient to keep the cycle functioning as the continuously substance consuming step involving the synthesis of citrate keeps the oxaloacetate levels in the cell extremely low, generally less than 1 micromolar, creating the conditions so that this last step of the Krebs cycle to be complete.
RegulationThe tricarboxylic acid cycle is largely regulated by substrate availability in the intracellular environment as well as by product inhibition. Moreover regulation is done by rate determining enzymes, which are, in this particular case of the TCA cycle, Citrate Synthase, Isocitrate Dehydrogenase and α-Ketoglutarate Dehydrogenase. As identifying a pathway’s rate-determining reactions demands the determination of the free energy of each reaction from the concentrations of its products and reactants, it is very hard to analyse the thermodynamics of the citric acid cycle as the concentrations of the intermediates involved are present in both mitochondria and cytosol in varying proportions. Nonetheless, if equilibrium between compartments is assumed, three reactions can be identified as displaced from their equilibrium points, may mean that these three reactions can be considered the rate-determining reactions of the TCA cycle.
Then, the TCA cycle is also regulated by the availability of NAD+ as there are 3 reactions that consume NAD+ and produce NADH. So, it would be reasonable to say that these reactions are regulated by NAD+ concentration. Moreover, four other reactions of the cycle are regulated through negative feedback loops by the concentration of NADH. Calcium ions also play a vitally important role in regulating the process by means of enhancing the reaction rates of the dehydrogenase reactions as it is an important cofactor in these reactions.
Nonetheless, maybe the most important regulators are oxaloacetate and Acetyl-CoA. Since the equilibrium of the reaction displaced so far to the left, discouraging the reaction to take place, it is essential that the concentrations of oxaloacetate are regulated so that the reaction can take place. In order for the reaction to be favoured, the reaction product concentrations in the cell have to be kept really low so as to promote the forward reaction and not the backwards one as predicted by thermo dynamical calculations. This is why the concentration of citrate is kept really low in cells, less than 1 microb Molar.
One of the most toxic small organic substances there is, fluoroacetate, is highly toxic not due to its native structure but due to the metabolic products in the body upon digestion. Since acetates are digested using an enzyme called acetate thiokinase, fluoroacetyl-CoA will be thus generated, this being further metabolized by citrate synthase intro fluorocitrate. The problem with this is that the C2 position is occupied by the fluoride atom and aconitase cannot catalyse the reactions in the cycle further-on. Since the fluorocitrate fits into the active site, we can thusly conclude that it’s a competitive inhibitor of citrate.
- E1 is also regulated as it comes in two forms: active (dephosphorylated), inactive (triphosphorylated). The enzyme which catalyzes this rather unusual phosphorylation by inactivation is Pyruvate Dehydrogenase Kinase, which generated three ADP and the phosphorylated enzyme at the cost of 3ATP. This enzyme is at its turn catalyzed by the PDC products, or by the acetylated E2 PDC enzyme. In contrast, it is inhibited by the PDC products (Pyruvate, NAD+, CoA). In reverse, the phosphorylation process is reversed by the activity of PD phosphatase, hydrolyzing the enzyme in three loci corresponding to the serine residues undergoing phosphorylation. The process is enhanced by the presense of Mg and Ca ions.
- The lipoic acid is covalently linked to a Lys residue in E2 and the lipoyllysyl arms transfer the metabolic intermediates between enzyme subunits.
- Arsenic, in the form of inorganic arsenite (H2AsO3-) or the organic arsenicals (R-As=O) are toxic as they covalently bind to the dilydrolipoamide freeing water and blocking the function of PDC. With this view, organic arsenicals are more toxic to bacteria, reason for which it was used as an antibiotic in the early 20th century.