Glycolysis

GLYCOLYSIS, glucolysis (from Greek glycos—sweet and lysis—breaking down), an enzymatic process of carbohydrate breakdown with their conversion into lactic acid. Liebig, who first established the presence of lactic acid in the body and isolated it in pure form, already pointed to carbohydrates as the source of its formation. Remarkable is the brilliant foresight of Liebig, who noted insufficient oxygen supply to tissues as a condition for the formation of lactic acid in the body. Only in the last decades has it been experimentally proven that this condition is indeed necessary for normal tissues. Claude Bernard (Cl. Bernard), who observed the conversion of carbohydrates into lactic acid, characterized this process as enzymatic; he also discovered the disappearance of sugar in blood released from vessels. The latter process was studied in more detail by Lépine, who proposed the term "glycolysis." For a long time, this term was used specifically to denote the process of sugar breakdown in the blood, although it had long been discovered that significant amounts of lactic acid also accumulate in muscle, especially during tetanic contraction and in a state of rigor. At the present time, after it has been established that the ability to break down sugars into lactic acid is inherent in all animal cells, the term "glycolysis" is used in all cases where the indicated conversion takes place.

Chemistry of glycolysis. From the equation C₆H₁₂O₆ (hexose) = 2C₃H₆O₃ (lactic acid), representing the initial and final stages of glycolysis, it is evident that this process represents, in total, a simple cleavage of the molecule, not associated with the addition of water elements, as is the case with hydrolysis (see). Oxygen also does not participate in the reaction: glycolysis is a purely anaerobic process, being the main pathway of oxygen-free breakdown of carbohydrates in the animal cell, just as alcoholic and other types of fermentation are pathways of anaerobic sugar breakdown in the cells of lower microorganisms.

However, a comparison of the structural formulas of hexose and lactic acid shows that glycolysis is the result not of a simple rupture of the six-membered carbon chain of hexose, but is associated with significant intramolecular rearrangements:

[Chemical structural diagram]

sn, hexose lactic acid These rearrangements are apparently carried out by the movement or sequential addition and removal of water elements (H and OH). It is undoubtedly the case that the entire process does not occur in one stage, but through a series of intermediate stages. Despite the enormous number of studies devoted to the chemistry of anaerobic carbohydrate breakdown as one of the central problems of modern biochemistry, the pathway of this breakdown has not yet been elucidated, and there are as yet no finally established data regarding its intermediate stages. On the basis of all the experimental material currently available, the most probable intermediate products in G. can be considered to be two compounds: CH₂OH-CHOH-COOH (glyceraldehyde) or CH₃-CO-COOH (ketoaldehyde-methylglyoxal); its other tautomeric enol formula is: CH₃=C(OH)-COOH. Less likely, but possible, is the intermediate formation of an isomer of glyceraldehyde-dihydroxyacetone CH₂OH-CO-CH₂OH. The likelihood of glyceraldehyde and methylglyoxal forming as intermediate products in G. is supported by the fact that both of these compounds, when isolated in organs, tissues, and extracts from them, are easily converted into lactic acid. However, it has not yet been possible to isolate these compounds during glycolysis. For glyceraldehyde, the direct transition into lactic acid seems, on the basis of stereochemical considerations, rather unlikely; rather, it should be assumed that it first, losing water, passes into methylglyoxal. The latter is thus apparently an obligatory intermediate product. Its conversion into lactic acid easily occurs under the influence of the enzyme glyoxalase or keto-aldehyde-mutase, which is widely distributed in animal tissues; it represents an example of an intramolecular oxidation-reduction process—the keto group CO is reduced to CHOH, while the aldehyde group CHO is oxidized to the carboxyl COOH. Participation of phosphoric acid. In G., as in fermentation, an important condition for the cleavage of the hexose carbon chain is the preliminary attachment of phosphoric acid (phosphorylation) to the latter molecule, forming a complex ester—hexose phosphate. The formation of such hexosephosphates was proven by Embden, who discovered the so-called lactacidogen (hexosediphosphoric acid). According to modern concepts (Meyerhof), in G. first hexosemonophosphoric acid is formed, which decomposes with the formation of lactic acid, and the liberated phosphoric acid attaches to a second hexosephosphate molecule, forming hexosediphosphate; the latter in turn decomposes, but more slowly, into lactic and phosphoric acids. Phosphorylation in G. has been established with certainty in the blood; with a high degree of probability it should also be assumed for other animal tissues. The meaning of phosphorylation apparently lies in the fact that when phosphoric acid is attached, the bonds in the hexose molecule are weakened and the cleavage of the carbon chain becomes possible. Role of cellular structure. G., or rather, its known stages, are closely connected with the presence of a specific cellular structure—when the latter is destroyed (grinding, freezing and thawing of tissues, hemolysis) or when surface-active substances (in particular narcotics) that are adsorbed on the structural surfaces of the cell are present, G. is weakened or completely ceases. A number of data indicates that the presence of structure is necessary for the very first stages of G., specifically for converting the normal form of glucose (α, β) into a more reactive, 'alloomorphic' modification (neoglucose, γ-glucose, o-glucose), which differs in the arrangement of internal bonds in the molecule. A certain role in this transformation is attributed to insulin. The question of the role of structure cannot be considered finally resolved, since recently it has been possible to obtain from muscles extracts and preparations that completely lack cells and yet vigorously carry out glycolysis. Such extracts glycolyze most intensely when glycogen or products of starch breakdown (hexosans), which according to Pringsheim contain γ-glucose, are used as the substrate. Co-enzyme of G. Another important factor necessary for G. is the presence of a co-enzyme. When cells or tissues are repeatedly washed with water, physiological or Ringer's solution, some substance is removed without which G. cannot occur. This substance is thermostable, not destroyed by boiling. When added to washed tissue that has lost its glycolytic ability, it restores G. This co-enzyme, whose chemical nature has not yet been elucidated, is contained in yeast, muscles, and other tissues. It is particularly abundant in rapidly growing cancerous tumors. It participates in the initial stages of glycolysis, apparently being necessary for the phosphorylation of the carbohydrate. The entire process of G. should be regarded as the result of a series of successive enzymatic transformations, each of which may be conditioned by a separate enzyme. Thus, it is very likely that here we are dealing not with a single 'glycolytic enzyme' but with some enzyme system. It is further necessary to bear in mind that all reactions occurring in G. are reversible and, depending on various conditions, can proceed in one direction or the other. Here we have a system of mobile chemical equilibria, and the final product of G. that we capture—lactic acid—represents a product of a certain temporary stabilization. Influence of external factors. Reaction of the medium. Like all enzymatic processes, glycolysis depends to the highest degree on the reaction of the medium. For G. in blood the optimal pH is 7.52-8.0; at pH 6.3 G. is already strongly weakened. For other tissues the optimum also lies between pH 7.0-8.0. The process of phosphorylation is especially sensitive to changes in the reaction of the medium; with even small shifts in [H+] it can proceed either toward synthesis (pH > 7.5) or toward cleavage (pH < 7.5). Temperature. At 0° G. completely stops, reaching a maximum at 37-40°; heating to 56° stops G. The enzyme complex is especially sensitive to increased temperature when it is separated from the cell; under these conditions it is already destroyed at room temperature, and to obtain active extracts it is necessary to carry out extraction at 0° or even -2°. Ions. Most cations have no noticeable effect on G., with the exception of Ca, which has an inhibitory effect; to a lesser extent Mg has a similar effect. Among anions, F acts extremely strongly; fluorides (salts of hydrofluoric acid) at a concentration of 1/500 completely stop the formation of lactic acid in muscle. Other anions are arranged according to their effect in the Hofmeister series (see). Specific activators of G. are bicarbonates and phosphates; their effect is manifested independently of their buffer properties. In the absence of these ions G. apparently cannot occur at all. Salts of arsenious acid (somewhat weaker—arsenic acid) strongly increase the intensity of G.: at a concentration of 0.4% in muscle glycolysis can increase by 100%. Recently, the strong activating effect of sulfates on G. has been noted. G. is not inhibited by hydrocyanic acid; under aerobic conditions HCN even enhances G., but this effect is not direct but indirect: by inhibiting oxidative processes, HCN promotes the accumulation of lactic acid (cf. below). Among pharmacological substances, the inhibitory effect of narcotics has already been noted. It should be noted that this effect varies greatly for different tissues, and for example in muscle at certain concentrations an increase in G. even occurs. According to Meyerhof, caffeine significantly enhances G. All attempts to elucidate the effect of the basic activator of carbohydrate metabolism—insulin—on G. have so far been unsuccessful. In vitro its effect on G. (i.e., on the anaerobic breakdown of sugar) cannot be established, whereas on the aerobic breakdown of sugar insulin, according to the experiments of Neuberg and Gottschalk, exerts a very pronounced activating effect even in vitro. The glycolytic ability of tissues of depancreatized animals shows no significant deviations from normal. From the pancreas it is possible to isolate a substance that strongly inhibits G. in muscle; this substance has absolutely no effect on the glycolytic ability of malignant tumors, from which it can be concluded that in different tissues G. has certain specific peculiarities. Energy significance of glycolysis and its connection with respiration. The conversion of hexose into lactic acid is an exothermic process associated with the release of energy: when 1 g of glucose is broken down, about 130 small calories are released. This energy is either used for endothermic processes associated with cell metabolism, or converted into mechanical work, or finally given off in the form of heat.

Despite the fact that when sugar is converted into lactic acid, significantly less energy is released than when it is oxidized to CO₂ and H₂O, as is the case in respiration (about 4,000 small calories per 1 g), G. still plays a primary role in the energy exchange of the living cell. This role manifests not only under anaerobic conditions, when G. is the only source of energy for the cell, but also in the presence of O₂, i.e., when oxidative processes are possible. In the latter case, in normal resting tissues and cells, since they possess the ability for oxidative exchange, the formation of lactic acid from sugar is not observed. However, in reality, even under aerobic conditions, one always deals with G., but it remains hidden, since the lactic acid formed (or the intermediate products directly preceding it, for example, methylglyoxal) is immediately converted back into carbohydrate (resynthesized). The energy for this endothermic reaction is provided by the oxidative processes. Thus, under aerobic conditions, there is a special cycle, which can be represented by the scheme (Meyerhof): carbohydrate _&lt;

-*" lactic acid. This cycle is called the Meyerhof reaction. Phase I is exothermic, it proceeds spontaneously with the release of energy; Phase II is endothermic and occurs due to simultaneously occurring oxidative processes. If the latter are absent (anaerobiosis, inhibition of respiration by hydrocyanic acid, inability of the cell to carry out oxidative processes), then Phase II is also absent, resynthesis is impossible, lactic acid accumulates, and G. is detected ⁴ directly. The indicated connection between respiration and anaerobic breakdown, foreseen by Pasteur, is, as established by Meyerhof and Warburg, common to all studied cells and tissues and represents one of the basic principles of cellular energy exchange. It has not yet been clarified what exactly serves as the substrate for oxidation in Phase II of the Meyerhof reaction—whether part of the formed lactic acid or the carbohydrate as such; the latter is more probable. In any case, it is established that the energy released during the oxidation of 1 molecule of lactic acid or an equivalent amount of carbohydrate is sufficient to cause the reverse conversion (resynthesis) into carbohydrate of 4-6 molecules of lactic acid. The ratio of the number of molecules of lactic acid resynthesized at the expense of the oxidation of one molecule of it is called the Meyerhof coefficient. This coefficient, determined for a number of different cells and tissues, fluctuates within relatively narrow limits, between 3 and 6. It also represents the coefficient of utilization by the cell of the energy of oxidative processes. The biological meaning of the Meyerhof reaction will become clearer if one uses a somewhat crude but vivid analogy: if one compares the cell to a clock mechanism, then the movement of individual wheels, the pendulum, the striking of the clock will correspond to the various energetic manifestations of the cell's life processes. The driving force of these processes, analogous to the spring in a clock, in the cell is G. The loss of carbohydrate and the accumulation of lactic acid corresponds to the weakening of the spring. But lactic acid in the cell, like the unwound spring in a clock, is not a completely spent material, and due to external energy (oxidation, winding the clock) it is again converted into the original, energy-rich state. Thus, the primary source of energy for the cell is G., while the energy of oxidative processes is used only indirectly, through resynthesis. The indicated exchange mechanism is essentially important for the cell in two respects: first, the same amount of substance participates in the G. reactions serving as a source of energy not once, as would be the case in the absence of resynthesis, but 4-6 times, which manifests the extreme frugality of the organism; on the other hand, even obligately aerobic cells become less dependent on the oxygen supply at any given moment, since oxidative processes are necessary only when an excess of lactic acid accumulates and the carbohydrate reserve correspondingly decreases. In normal tissues, the oxidative processes (respiration) are so intense that the energy they provide with more than enough suffices for the resynthesis of all the lactic acid formed, so that the latter is not detected at all (aerobic G. here—somewhat unhappily—is taken =0). The appearance of lactic acid under aerobic conditions (aerobic G.) becomes possible in all those cases when the energy of respiration is insufficient for resynthesis. The table below compares the values of anaerobic and aerobic G. and respiration of various tissues (concerning the absolute value of the table numbers—see below). From it it is seen that rapidly growing tissues (tumors, embryo) have vigorous G.; since their respiration is also very intense, it suffices for resynthesis, and under aerobic conditions practically no lactic acid is formed. In resting, non-growing tissues G. is significantly weaker (an exception is the retina and gray matter), and respiration also with more than enough suffices to compensate for G. In the anucleate erythrocytes of mammals, cells practically devoid of oxidative exchange, anaerobic and aerobic G. differ little in magnitude. This explains why G. was first discovered in the blood. A sharp difference from normal tissues is presented by malignant tumors, where along with very vigorous G. there is only weak respiration. Correspondingly to this, cancerous and sarcomatous cells, even in the presence of oxygen, form significant amounts of lactic acid (intense aerobic G.). The intensity of G. and respiration of various tissues and cells is expressed in the following figures (concerning their absolute value see below): Anaerobic G. Respiration Aerobic G. Retina...........

30,7

31 7,2

-21 5,1

14 Human sarcoma . . .

20,6

ю

1,1 Gray matter . .

19,1

10,7

2,5 Dog tumors

4,6 Tonsils (human)....

12,8

5,1

8,2 5,8

3,5

11,6

3,3

0,0 Pancreas . . .

3,4 4,6

0,0 Thyroid gland ....

2,1

0,0 Erythrocytes (rabbit) ....

0,43

0,4-0,6

20 9

2,0 Blood platelets ....

25 6

1,0 Lens (adult animal)

1 1

0,0

27 8

17,0

5 6

1,0

Physiological significance of glycolysis. Back in the last century, Hoppe-Seyler suggested that the formation of lactic acid under anaerobic conditions is a physiological function inherent to all living protoplasm. The work of recent years, especially that of the Meyerhof and Warburg laboratories, has fully confirmed this. The significance of glycolysis in muscle contraction has been clarified with the greatest certainty. Here, lactic acid, which is formed in response to a nerve impulse or stimulation from glycogen through its hydrolytic cleavage and subsequent glycolysis, serves as the main driving force, and it is precisely this, through the mediation of various physical and colloid-chemical mechanisms, that determines the entire process of muscle contraction. Thus, glycolysis turns out to be a process underlying one of the most important and characteristic functions of the animal organism—the ability to perform mechanical work. Another fundamental function of living matter—the ability to grow—is likewise most closely connected with glycolysis, since the intensity of the latter is especially high precisely in growing tissues (tumors, developing embryos), and the transition of cells from rest to growth and reproduction is accompanied by a sharp increase in the intensity of glycolysis. Warburg believes that "without glycolysis there is no growth" and sees in glycolysis the main driving force at the expense of which the work associated with growth and reproduction is performed. The extraordinary intensity of glycolysis in such highly differentiated tissues as the retina and the cerebral cortex suggests that their functions are in one way or another connected with glycolysis. Glycolysis in blood is associated exclusively with the formed elements, with the erythrocytes playing the main role. The indications of previous authors that leukocytes glycolyze 100 times more energetically than erythrocytes are hardly correct. Judging by recent work, under aerobic conditions, leukocytes glycolyze only 15 times more strongly than red blood cells, and only under anaerobiosis does the intensity of glycolysis in them become 40 times higher than in erythrocytes. Corresponding to the quantitative ratios of both types of cells in the blood, the leukocytes account for only a very insignificant part of the total glycolysis. Bizzozero platelets also possess glycolytic ability (Fleischmann, Kubowitz, Fujita). Destruction of the erythrocyte structure (hemolysis) terminates glycolysis; however, the formation of lactic acid from the presumed intermediate products of glycolysis—from glyceraldehyde and dioxyacetone—occurs even in hemolyzed blood, which is further proof that the cellular structure is necessary only for the first stages of glycolysis. The intensity of glycolysis in the blood of various animals (see table below) is linked to the varying permeability of erythrocytes to glucose. It is highly probable, however, that this also depends on a number of other factors. The intensity of glycolysis in the blood of different animals (loss of sugar in mg per 100 cubic cm of blood per 1 hour at 37°) is expressed by the following figures: [Table data omitted in source text]

14.8

22.3

1.5-0.0

- Nucleated erythrocytes of birds, which possess energetic respiration, practically do not form lactic acid under aerobic conditions, whereas under anaerobic conditions they glycolyze approximately the same as the non-nucleated erythrocytes of mammals. In turn, the latter, in the presence of methylene blue, which significantly increases oxidative processes, also almost cease to form lactic acid (Barron, Harrop). Glycolysis in the blood is undoubtedly connected with phosphorylation and is accompanied by the formation of relatively stable organic compounds of phosphoric acid [apparently mainly diphosphoglyceric acid (Jost)].—Glycolysis in pathological states. Characteristic changes in glycolytic ability have been established with certainty so far only in malignant neoplasms (see Tumors). Numerous studies aimed at detecting changes in the intensity of glycolysis depending on various pathological conditions have not yet yielded definite results. In particular, in diabetes, despite a sharp disturbance of general carbohydrate metabolism, it has not been possible to establish any regular changes in glycolysis in the blood; the weakening of glycolysis noted by some authors in diabetic coma, if real, is in all probability due to a change in pH. Indications that glycolysis increases during fatigue, in nephritis, is weakened in old age, in tuberculosis, etc., need thorough verification. Methodology for studying glycolysis. Corresponding to the very definition of glycolysis, a correct idea of glycolysis can be given only by a simultaneous accounting of the loss of sugar and the appearing lactic acid. Limiting oneself to the determination of only one of these values is unreliable, since sugar can be partially oxidized or converted into some other compounds besides lactic acid; on the other hand, the latter can also be formed from other compounds, e.g., from amino acids. It is, of course, very important to take into account the intensity of respiration. Warburg has developed a very precise, elegant, and convenient manometric method for accounting for glycolysis: the experiment is conducted in special small vessels connected to manometers; the formed lactic acid displaces carbon dioxide from the bicarbonates of the Ringer's solution in which the tissue under study is suspended; by the change in pressure read on the manometer, one can judge the amount of lactic acid. In relation to this method, Warburg proposed the following designations for the quantitative characterization of glycolysis: Q^N2_M—the coefficient of anaerobic glycolysis; the sign M indicates that the coefficient refers to lactic acid (Milchsaure); N₂ indicates that the experiment is conducted in an atmosphere of nitrogen (anaerobiosis). The corresponding coefficients for aerobic glycolysis are denoted by the symbol Q^O2_M, and for respiration—Q_O2. These coefficients for glycolysis denote the amount of lactic acid (expressed in cubic mm of CO₂ displaced by it from bicarbonate) formed in 1 hour by a given tissue, calculated per 1 mg of its dry matter. Q_O2 denotes the amount of oxygen in cubic mm consumed in 1 hour by the tissue, also calculated per 1 mg of dry matter. 1 cubic mm of CO₂ is equivalent to 0.00402 mg of lactic acid, so that by multiplying the Q_M values by 0.00402, one can express the amount of lactic acid directly in mg.