In this article we will discuss about the formation of ATP in anaerobic cells.
We know that the formation of ATP in the respiratory chain is because it is the most common process in living organisms. The strict anaerobes, i.e. species which can live only in the absence of oxygen (in the ground or mud, for example) are comparatively rare.
On the contrary, there are some facultative anaerobes, organisms which preferentially utilize oxygen to oxidize their nutritive substances when oxygen is present but which can also live in absence of oxygen, then drawing the energy they require from fermentation processes.
This capacity of conserving the energy of food in anaerobiosis (production of ATP during fermentation) is found not only in some microorganisms but also in numerous more advanced organisms, and the formation of ATP during the fermentation of glucose is a process of energy conservation which has reached man; as we will see below, the anaerobic degradation of glucose precedes its complete oxidation by the Krebs cycle and the respiratory chain.
Various substances (sugars, notably glucose, fatty acids, amino acids) can be utilized by bacteria in anaerobiosis, which also helps in identifying or classifying these bacteria. Furthermore, these bacteria — even when they utilize glucose for example — can also be distinguished according to the mode of fermentation; some degrade glucose into ethanol, others into acetone, butanol, butyric acid, propionic acid or lactic acid.
But in a large number of cases, glucose is split into 2 fragments, of which one is oxidized by the other, and this oxidation- reduction process liberates energy, a part of which is stored in the form of ATP. We will study here only the fermentation of glucose to lactic acid, a mechanism found not only in certain bacteria (e.g., of the genus Lactobacillus), but also in higher animals (anaerobic glycolysis).
Energy Yield of Lactic Fermentation:
The transformation of glucose into lactic acid corresponds to the following overall equation:
In reality, in living cells, this cleavage of glucose implies a dozen of reactions and first the utilization of 2 ATP for the formation of glucose-6-phosphate and fructose-1,6-bisphosphate, then the formation of 4 ATP, which gives the following overall equation:
On the basis of ∆G0 values and admitting that the formation of one ATP requires at least 7 kcal/mole, one can calculate a minimum energy conservation efficiency of about 27% (2 x 7 x 100/52) with reference to the energy liberated by the cleavage of glucose into lactic acid; but if this yield is expressed with reference to the 686 kcal which can be liberated by the sudden oxidation of glucose, then it would be 14 X 100/686 # 2%, i.e. a very low yield compared to the 40% obtained in aerobiosis when glycolysis is followed by the Krebs cycles and by electron transfer in the respiratory chain.
It is quite clear that in anaerobiosis, only a very small part of the total energy of the glucose molecule can be “recovered” and consequently, to accomplish an identical work, anaerobic cells must consume much more (up to 20 times more) glucose per unit time than the aerobic cells.
Glycolysis therefore appears to be a more primitive, less elaborate means of storing energy in the form of ATP; this is further confirmed by the fact that the enzymes (about ten) catalysing the various steps are in free state in the soluble part of the cellular cytoplasm, and not grouped in pluri-enzymatic systems of complex structure like the enzymes catalyzing the reactions of respiration and photosynthesis in mitochondria and chloroplasts.
Mechanisms of Energy Conservation. Substrate-Related Phosphorylation:
Figure 4-35 which summarizes the various steps of glycolysis, one observes that paradoxically the first steps of this process (which must lead to the formation of ATP) consume ATP: these steps are the phosphorylation of glucose and that of fructose-6-phosphate.
But then, for each C3 fragment there are 2 steps of phosphorylation of ADP into ATP and therefore, 4 ATP are formed as one molecule of glucose gives 2 C3 fragments. Finally, 2 ATP are consumed and 4 ATP formed, which is conform to the gain of 2 ATP indicated by the overall reaction given in the previous paragraph. We will now see the mechanisms by which these ATP molecules are formed during the glycolysis.
In addition to phosphorylations which take place along the respiratory chain and to photosynthetic phosphorylations, there are substrate-related phosphorylations; two examples are given below.
A. Formation of ATP during the oxidation of glyceraldehyde 3-phosphate into 3-phosphoglyceric acid:
The oxidation of an aldehyde to acid:
R-CHO + H2O → R-COOH + 2H
is an exergonic reaction (∆G0 # -7 kcal/mole). In the cells, this energy is not necessarily dissipated as heat; it can be utilized for the endergonic reaction of ATP formation:
ADP + Pi → ATP + H2O (∆G0 # +7 kcal/mole)
The energy produced by the oxidation of aldehyde is then stored in the ATP formed and one can write an overall reaction, the free energy change of which is practically nil:
R-CHO + ADP + Pi → R-COOH + 2H + ATP (∆G0#0)
This is a process involving 2 enzymes — a dehydrogenase, then a kinase — with intermediate formation of an acyl-phosphate, a high energy potential compound (see table) which can liberate energy of the order of 10 kcal/mole during its hydrolysis.
This common intermediate is, on the one hand, the oxidation product of 3-phosphoglyceraldehyde (first reaction), and on the other hand, the donor of the phosphate group to ADP to form ATP (second reaction), as shown by figure 3-10. The free energy of hydrolysis of this compound is considerable because of the high electron density of the anhydride bond between the phosphate and the carboxyl, and of the proximity of the 2 phosphate groups with their double negative charge.
One can see that during the dehydrogenation of 3-phosphoglyceraldehyde there is formation of NADH + H+. When the respiratory chain functions, the transfer of electrons from NADH + H+ to oxygen enables – via the shuttles of the reducing power – the formation of 3 ATP, and since one glucose molecule gives 2 C3 fragments, 6 additional ATP can be formed when glycolysis is followed by the aerobic phase, but not during the strict anaerobic glycolysis.
B. Formation of ATP during the conversion of 2-phosphoglyceric acid into pyruvic acid:
It was mentioned above that — in glycolysis as well as in the respiratory chain — energy is stored in the form of ATP during the oxidation-reduction reactions. But, on examining 2-phosphoglyceric acid and pyruvic acid, it is not evident that the passage from one of these compounds to the other implies such a process.
In fact, the study of the detailed mechanism of this transformation (fig. 4-29) shows a displacement of electrons within the molecule which can be regarded as an intramolecular oxidation-reduction. There is here also, formation of an intermediate compound — phosphoenol-pyruvic acid — having a high energetic potential due to high electron concentration around the carbon 2, partly due to the formation of a double bond (2 pairs of electrons) and also to a decrease of distances and bond angles around this carbon.
This compound has a free energy of hydrolysis of the order of 12.8 kcal/mole (see table) so that, on yielding its phosphate group, it allows the formation of one molecule of ATP (see fig. 3-11).
Let us consider the second step of this transformation: since the hydrolysis of phosphoenolpyruvate has a AG0 # -12.8 kcal/mole and the formation of ATP, a ∆G0 # + 7 kcal/mole, the ∆G0 of the reaction phosphoenol-pyruvate + ADP → pyruvate + ATP is of the order of -5 to – 6 kcal/mole; if we take an average value of-5.45 kcal/mole and refer to the table below, we find that this value corresponds to a log K = + 4, i.e. K = 10 000; as we know, this value means that the equilibrium of the reaction will be reached when practically all the phosphoenolpyruvate and ADP will be transformed, in other words, this reaction is almost total.
In practice, one often adds phosphoenolpyruvate and pyruvate-kinase as ATP generating mixture in cell-free systems while studying in vitro endergonic reactions (synthesis of proteins for example) which could no longer take place if ATP were hydrolysed to ADP and happened to be lacking in the reaction mixture.
In the Krebs cycle, there is also a substrate-related phosphorylation.
