cofactor of the dehydrogenation. At this stage, it is in its oxidized form; nicotinamide is the reactive part of the molecule (Figure 2.3). Simultaneously, an energy‐rich phosphate ester bond is established between the oxidized carbon of the substrate and the inorganic phosphate. NAD+ accepts two electrons and a hydrogen atom lost by the oxidized substrate. Next, phosphoglycerate kinase catalyzes the transfer of the phosphoryl group of the acylphosphate from 1,3‐BPG to ADP; and 3‐phosphoglycerate and ATP are formed.
The last phase of glycolysis transforms 3‐phosphoglycerate into pyruvate. Phosphoglyceromutase catalyzes the conversion of 3‐phosphoglycerate into 2‐phosphoglycerate. Enolase catalyzes the dehydration of the latter, forming phosphoenolpyruvate. This compound has a high phosphoryl group transfer potential. By phosphorylation of ADP, pyruvic acid and ATP are formed; pyruvate kinase catalyzes this reaction. In this manner, glycolysis creates four ATP molecules. Two are immediately used to activate a new hexose molecule and the net gain of glycolysis is therefore two ATP molecules per molecule of hexose metabolized. This stage marks the end of the common trunk of glycolysis, which differentiates between alcoholic fermentation, glyceropyruvic fermentation, and respiration.
FIGURE 2.2 Glycolysis and alcoholic fermentation pathway.
FIGURE 2.3 (a) Structure of nicotinamide adenine dinucleotide in the oxidized form (NAD+). (b) Equilibrium reaction between the oxidized (NAD+) and reduced (NADH) forms.
2.2.2 Alcoholic Fermentation
The reducing power of NADH produced by glycolysis must be transferred to an electron acceptor in order to regenerate NAD+. In alcoholic fermentation, it is not pyruvate but rather acetaldehyde, its decarboxylation product, that serves as the terminal electron acceptor. With respect to glycolysis, alcoholic fermentation contains two additional enzymatic reactions.
The first decarboxylates pyruvic acid, catalyzed by pyruvate decarboxylase (PDC). The cofactor is thiamine pyrophosphate (TPP) (Figure 2.4). TPP and pyruvate form an intermediate compound. More precisely, the carbon atom located between the nitrogen and the sulfur of the thiazole ring of TPP is ionized. It forms a carbanion, which readily combines with the pyruvate carbonyl group. The second step reduces acetaldehyde into ethanol by NADH. This reaction is catalyzed by alcohol dehydrogenase, whose active site contains a Zn2+ ion.
Saccharomyces cerevisiae PDC comprises two isoenzymes: a major form, PDC1, representing 80% of the decarboxylase activity, and a minor form, PDC5, whose function remains uncertain.
From an energy viewpoint, glycolysis followed by alcoholic fermentation therefore supplies the yeast with two molecules of ATP per molecule of glucose degraded or 14.6 biologically usable kcal per mole of glucose fermented. From a thermodynamic viewpoint, the change in free energy during the degradation of a mole of glucose into ethanol and CO2 is −40 kcal. The difference (25.4 kcal) is dissipated in the form of heat.
FIGURE 2.4 Structure of thiamine pyrophosphate (TPP).
2.2.3 Glyceropyruvic Fermentation
In the presence of sulfite (Neuberg, 1946), the fermentation of glucose by yeasts produces equivalent quantities of glycerol, carbon dioxide, and acetaldehyde in its bisulfite form. This glyceropyruvic fermentation takes place in the following manner. Since the sulfite‐bound acetaldehyde cannot be reduced into ethanol, dihydroxyacetone phosphate becomes the terminal acceptor of electrons from the oxidation of glyceraldehyde 3‐phosphate, which is then reduced to glycerol 3‐phosphate. The latter is dephosphorylated into glycerol. This mechanism was used for the industrial production of glycerol. In this fermentation, only two molecules of ATP are produced for every molecule of hexose degraded. ATP is required to activate the glucose in the first step of glycolysis (Figure 2.5). Glyceropyruvicfermentation, whose net gain in ATP is nil, does not furnish biologically assimilable energy for yeasts.
Glyceropyruvic fermentation does not occur solely in a highly sulfited environment. At the beginning of alcoholic fermentation of grape must, the inoculum consists of yeasts initially grown in the presence of oxygen. Their PDC and alcohol dehydrogenase are weakly expressed. As a result, acetaldehyde accumulation is limited. The reoxidation of NADH therefore does not involve acetaldehyde, but rather dihydroxyacetone. Glycerol, pyruvate, and some secondary fermentation products are formed. These secondary products are pyruvate derivatives—including, but not limited to, succinate and diacetyl.
2.2.4 Respiration
When sugar is used by the respiratory pathway, pyruvic acid (originating from glycolysis) undergoes an oxidative decarboxylation in the presence of coenzyme A (CoA) (Figure 2.6) and NAD+. This process generates carbon dioxide, NADH and, acetyl‐CoA:
The enzymatic complex of pyruvate dehydrogenase catalyzes this reaction. It takes place inside the mitochondria. TPP, lipoamide, and flavin adenine dinucleotide (FAD) participate in this reaction and serve as catalytic cofactors.
The acetyl unit coming from pyruvate is activated in the form of acetyl‐CoA. The reactions of the citric acid cycle, also called the tricarboxylic acid cycle or Krebs cycle (Figure 2.7), completely oxidize the acetyl‐CoA into CO2. These reactions also occur in the mitochondria.
This cycle begins with the condensation of a two‐carbon acetyl unit with a four‐carbon compound, oxaloacetate, to produce a tricarboxylic acid with six carbon atoms: citric acid. Four oxidation–reduction reactions regenerate the oxaloacetate. The oxidative pathway involves decarboxylation of isocitrate, an isomer of citrate, into α‐ketoglutarate. Isocitrate dehydrogenase catalyzes this reaction. A five‐carbon compound, α‐ketoglutarate, undergoes an oxidative decarboxylation to become succinate, catalyzed by α‐ketoglutarate dehydrogenase. In these two reactions, NAD+ is the hydrogen acceptor. Fumarate dehydrogenase is responsible for the reduction of succinate into fumarate. FAD is the hydrogen acceptor (Figure 2.8). Finally, fumarate is hydrated into L‐malate. The latter is reduced into oxaloacetate by malate dehydrogenase. In this case, the NAD+ is the electron acceptor once again.
