Pyruvate, the product of glycolysis, represents an important junction point in carbohydrate catabolism (Fig. 14-3). Under aerobic conditions pyruvate is oxidized to acetate, which enters the citric acid cycle (Chapter 15) and is oxidized to CO2 and H2O. The NADH formed by the dehydrogenation of glyceraldehyde-3-phosphate is reoxidized to NAD+ by passage of its electrons to O2 in the process of mitochondrial respiration (Chapter 18). However, under anaerobic conditions (as in very active skeletal muscles, in submerged plants, or in lactic acid bacteria, for example), NADH generated by glycolysis cannot be reoxidized by O2. Failure to regenerate NAD+ would leave the cell with no electron acceptor for the oxidation of glyceraldehyde-3-phosphate, and the energy-yielding reactions of glycolysis would stop. NAD+ must therefore be regenerated by some other reaction.
The earliest cells to arise during evolution lived in an atmosphere almost devoid of oxygen and had to develop strategies for carrying out glycolysis under anaerobic conditions. Most modern organisms have retained the ability to continually regenerate NAD+ during anaerobic glycolysis by transferring electrons from NADH to form a reduced end product such as lactate or ethanol.
Pyruvate Is the Terminal Electron Acceptor in Lactic Acid Fermentation
When animal tissues cannot be supplied with sufiicient oxygen to support aerobic oxidation of the pyruvate and NADH produced in glycolysis, NAD+ is regenerated from NADH by the reduction of pyruvate to lactate. Certain other tissues and cell types (retina, brain, erythrocytes) also produce lactate from glucose under aerobic conditions; lactate is a major product of erythrocyte metabolism. The reduction of pyruvate is catalyzed by lactate dehydrogenase, which forms the L-isomer of lactic acid (lactate at pH 7). The overall equilibrium of this reaction strongly favors lactate formation, as shown by the large negative standard free-energy change. In glycolysis, dehydrogenation of the two molecules of glyceraldehyde-3-phosphate derived from each molecule of glucose converts two molecules of NAD+ to two of NADH. Because the reduction of two molecules of pyruvate to two of lactate regenerates two molecules of NAD+, the overall process is balanced and can continue indefinitely: one molecule of glucose is converted to two of lactate, with the generation of two ATP molecules from one of glucose, and NAD+ and NADH are continuously interconverted with no net gain or loss in the amount of either.
Although there are two oxidation-reduction steps as glucose is converted into lactate, there is no net change in the oxidation state of carbon; in glucose (C6H12O6) and lactic acid (C3H6O3), the H:C ratio is the same. Nevertheless, some of the energy of the glucose molecule has been extracted by its conversion to lactate, enough to give a net yield of two molecules of ATP for every one of glucose consumed. The lactate formed by active muscles of vertebrate animals can be recycled; it is carried in the blood to the liver where it is converted into glucose during the recovery from strenuous muscle activity (Box 14-1).
Many microorganisms ferment glucose and other hexoses to lactate. Certain lactobacilli and streptococci, for example, ferment the lactose in milk to lactic acid. The dissociation of lactic acid to lactate and H+ in the fermentation mixture lowers the pH, denaturing casein and other milk proteins and causing them to precipitate. Under the correct conditions, the resultant curdling produces cheese or yogurt, depending on which microorganism is involved
Ethanol Is the Reduced Product in Alcohol FermentationYeast and other microorganisms ferment glucose to ethanol and CO2, rather than to lactate. Glucose is converted to pyruvate by glycolysis, and the pyruvate is converted to ethanol and CO2 in a two-step process. In the first step, pyruvate undergoes decarboxylation in the irreversible reaction catalyzed by pyruvate decarboxylase. This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate. Pyruvate decarboxylase requires Mg2+ and has a tightly bound coenzyme, thiamine pyrophosphate, discussed in more detail below.
In the second step, acetaldehyde is reduced to ethanol, with NADH derived from glyceraldehyde-3-phosphate dehydrogenation furnishing the reducing power, through the action of alcohol dehydrogenase. Ethanol and CO2, instead of lactate, are thus the end products of alcohol fermentation. The overall equation of alcohol fermentation is
Glucose + 2ADP + 2Pi ____________2 ethanol + 2CO2 + 2ATP + 2H2OAs in lactic acid fermentation, there is no net change in the ratio of hydrogen to carbon atoms when glucose (H : C ratio = 12/6 = 2) is fermented to two ethanol and two CO2 (combined H : C ratio = 12/6 = 2). In all fermentations, the H : C ratio of the reactants and products remains the same.
Pyruvate decarboxylase is characteristically present in brewer's and baker's yeast and in all other organisms that promote alcohol fermentation, including some plants. The CO2 produced by pyruvate decarboxylation in brewer's yeast is responsible for the characteristic carbonation of champagne. The ancient art of brewing beer involves a number of enzymatic processes in addition to the reactions of alcohol fermentation (Box 14-2). In baking, CO2 released by pyruvate decarboxylase when yeast is mixed with a fermentable sugar causes dough to rise. The enzyme is absent in the tissues of vertebrate animals and in other organisms, such as the lactic acid bacteria, that carry out lactic acid fermentation.
Alcohol dehydrogenase is present in many organisms that metabolize alcohol, including humans. In human liver it brings about the oxidation of ethanol, either ingested or produced by intestinal microorganisms, with the concomitant reduction of NAD+ to NADH.
Thiamine Pyrophosphate Carries "Active Aldehyde" GroupsThe pyruvate decarboxylase reaction in alcohol fermentation represents our first encounter with thiamine pyrophosphate (TPP) (Fig. 14-9), a coenzyme derived from vitamin Bl. The absence of vitamin Bl in the human diet leads to the condition known as beriberi, characterized by an accumulation of body fluids (swelling), pain, paralysis, and ultimately death .
Thiamine pyrophosphate plays an important role in the cleavage of bonds adjacent to a carbonyl group (such as the decarboxylation of α-keto acids) and in chemical rearrangements involving transfer of an activated aldehyde group from one carbon atom to another (Table 14-1). The functional part of thiamine pyrophosphate is the thiazolium ring (Fig. 14-9a). The proton at C-2 of the ring is relatively acidic, and loss of this acidic proton produces a carbanion that is the active species in TPP-dependent reactions (Fig. 14-9c). This carbanion readily adds to carbonyl groups, and the thiazolium ring is thereby positioned to act as an "electron sink" that greatly facilitates reactions such as the decarboxylation catalyzed by pyruvate decarboxylase.Microbial Fermentations Yield Other End Products of Commercial Value
Although lactate and ethanol are common products of microbial fermentations, they are by no means the only possible ones. In 1910 Chaim Weizmann (later to become the first president of Israel) discovered that a bacterium, Clostridium acetobutyricum, ferments starch to butanol and acetone. This discovery opened the field of industrial fermentations, in which some readily available material rich in carbohydrate (corn starch or molasses, for example) is supplied to a pure culture of a specific microorganism, which ferments it into a product of greater value. The methanol used to make "gasohol" is produced by microbial fermentation, as are formic, acetic, propionic, butyric, and succinic acids, glycerol, isopropanol, butanol, and butanediol. Fermentations such as these are generally carried out in huge, closed vats in which temperature and access to air are adjusted to favor the multiplication of the desired microorganism and to exclude contaminating organisms (Fig. 14-10). The beauty of industrial fermentations is that complicated, multistep chemical transformations are carried out in high yields and with few side products by chemical factories that reproduce themselves-microbial cells. In some cases it is possible to immobilize the cells in an inert support, to pass the starting material continuously through a bed of immobilized cells, and to collect the desired product in the effluent: an engineer's dream!