In this article we will discuss about the functions of the Krebs cycle, explained with the help of diagrams.
Krebs made an outstanding contribution to the discovery of the operating mechanisms of this cycle which is also called tricarboxylic cycle or citric cycle, owing to the participation of various tricarboxylic acids, including citric acid.
Before studying this cycle — which will take up acetyl coenzyme A, whose formation was explained above — it would be useful to place it correctly in the context of the entire cellular metabolism.
Since, acetyl coenzyme A is formed by oxidative decarboxylation of pyruvic acid which itself results from glucose by a series of transformations (glycolysis), the Krebs cycle — which permits the complete oxidation of glucose — can logically be included in the study of the metabolism of carbohydrates.
But one should bear in mind that the Krebs cycle is situated at the crossroads of metabolisms of carbohydrates, lipids and proteins and that it will achieve the complete oxidation to CO2 + H2O, of not only glucose, but also fatty acids whose catabolism leads to the formation of acetyl coenzyme A, and numerous amino acids whose metabolism leads either to the formation of pyruvic acid (and therefore of acetyl coenzyme A), or directly to the formation of one of the intermediates of the Krebs cycle (α-ketoglutaric, succinic, fumaric or oxaloacetic acid).
The steps of Krebs cycle are represented in fig. 4-38; we will now give a few details on the reactions which take place in this cycle.
Reactions of the Krebs Cycle:
In a first step, catalyzed by a condensing enzyme, or citrate-synthetase, the acetyl group is transferred from the acetyl coenzyme A to oxaloacetic acid to give citric acid, and coenzyme A is liberated.
Then, under the effect of one and the same enzyme, aconitase, citric acid is dehydrated to cis-aconitic acid (which is perhaps not liberated and would remain in that case linked to the enzyme), and rehydrated to isocitric acid; these three compounds coexist in equilibrium.
Then proceeds the oxidation of isocitric acid (with probable intermediate formation of oxalosuccinic acid), catalyzed by isocitrate dehydrogenase whose coenzyme is either NAD+ or NADP+, depending on the organism studied. The same enzyme catalyzes the decarboxylation of oxalosuccinic add to α-ketoglutaric acid.
Now, α-ketoglutaric acid undergoes an oxidative decarboxylation under the effect of an enzymatic complex (called α-keto-glutarate dehydrogenase), having as coenzymes, thiamine pyrophosphate, lipoic acid, coenzyme A, FAD and NAD+; the mechanisms are similar to those we studied in connection with the oxidative decarboxylation of pyruvic acid to acetyl coenzyme A (see fig. 4-36).
Succinyl coenzyme A is therefore obtained; it is very quickly split into succinic acid and coenzyme A, while the energy contained in the thio ester molecule is used for the synthesis of a molecule of ATP under the effect of succinate thiokinase:
Succinic acid is oxidized to fumaric acid, thanks to succinate dehydrogenase, the coenzyme of which is FAD. FADH2, will pass on its electrons to the electrons transport system but, the entry of electrons at this stage takes place beyond the first site of formation of ATP (which is, so to say, “skipped”), so that contrary to what happens with NAD+ or NADP+ dehydrogenases only 2 molecules (and not 3) of ATP will be formed per molecule of FADH2 reoxidized.
Fumaric acid is hydrated, thanks to fumarase, to malic acid and the latter is oxidized by the effect of malate dehydrogenase, a NAD+ enzyme, to oxaloacetic acid, thus closing the cycle. The oxaloacetic acid formed can again bind a new acetyl group which, in its turn, will be oxidized to CO2 + H2O after one turn of the cycle and so on.
The CO2 formed in the Krebs cycle can be used in various reactions (car- boxylations, synthesis of urea, biosynthesis of nucleotides) or eliminated by the lungs after transport by the blood stream.
Energy Balance:
To calculate the number of molecules of ATP formed per molecule of glucose, we must envisage, not one turn of the cycle (acetyl-CoA → 2CO2), but two turns, because 1 molecule of glucose gives 2 molecules of pyruvic acid (and therefore 2 molecules of acetyl-CoA).
This total of 38 ATP is considerable compared to the mere 2 ATP, which can be obtained in anaerobiosis. It must be indicated that the enzymes in Krebs cycle are mainly located in the mitochondria just as the enzymes involved in electron transport and oxidative phosphorylation, which most probably enhances the efficiency of coupling between the oxidation of acetyl coenzyme A and production of ATP.
However, whatever its importance, the energetic aspect of glucose oxidation must not overshadow the fact that glucose is not only a source of energy but also a source of carbon (glucose is often used as the sole source of carbon for growing bacteria).
In other words, from the products of glucose catabolism, the cells can synthesize a very wide variety of compounds: for example, fatty acids and steroids are synthesized from acetyl coenzyme A, and some amino acids (like alanine, glutamic acid, aspartic acid) are formed from the corresponding keto-acids (pyruvic, α-ketoglutaric and oxaloacetic acids respectively).
Formation and Decarboxylation of Oxaloacetic Acid:
If some intermediates of the Krebs cycle leave the latter because they are involved in other metabolic pathways, the quantity of oxaloacetic acid will decrease and the capacity of the cycle to accept acetyl coenzyme A will therefore decrease. To restore the oxidation capacity of the cycle, new molecules of oxaloacetic acid must be synthesized.
This can take place in several ways:
1) By deamination or transamination of either aspartic acid (which gives directly oxaloacetic acid) or glutamic acid (giving α-ketoglutaric acid which will be converted to oxaloacetic acid by the reactions of the cycle). This is a possible solution, provided that the same reactions in the reverse direction (in other words the necessity for the cells to form these amino acids) are not precisely the cause of the decrease in the quantity of oxaloacetic acid available for the oxidation of the acetyl coenzyme A.
2) By carboxylation reactions of various acids having 3 carbon atoms. We have already studied these CO2 fixation reactions leading to the formation of oxaloacetic acid, while discussing neoglucogenesis (see fig. 3-34);
We will only briefly mention them here:
i. Carboxylation of pyruvic acid to oxaloacetic acid catalyzed by pyruvate carboxylase
ii. Carboxylation of pyruvic acid to malic acid, catalyzed by “malic enzyme”, and oxidation of malic acid to oxaloacetic acid, catalyzed by malate dehydrogenase
iii. Carboxylation of phosphoenolpyruvic acid to oxaloacetic acid by phos- phoenolpyruvate carboxykinase.
The latter reaction, considered in the direction oxaloacetic acid → phosphoenolpyruvic acid, which was stated to allow the “reversibility” of glycolysis after the pyruvic acid is, in a first step, converted to oxaloacetic acid, therefore also allows the intermediates of the Krebs cycle to be converted into glucose.
The cycle cannot indeed turn in the opposite direction although most of its reactions are reversible (because the oxidative decarboxylation of α-ketoglutaric acid is not reversible), so that the main pathway through which the intermediates of the cycle (or compounds which can give these intermediates, like some glucoformer amino acids) can “return” to glucose, precisely passes through this decarboxylation reaction of oxaloacetic acid into phosphoenolpyruvic acid.