Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 50

This series of reactions, transforming glucose into pyruvate with the formation of ATP, constitutes a quasi‐universal pathway in biological systems. The elucidation of the different steps of glycolysis is intimately associated with the birth of modern biochemistry. The starting point was the fortuitous discovery by Hans and Eduard Buchner (1897), of the fermentation of sucrose by an acellular yeast extract. Studying possible therapeutic applications for their yeast extracts, the Buchners discovered that the sugar used to preserve their yeast extract was rapidly fermented into alcohol. Several years later, Harden and Young demonstrated that inorganic phosphate must be added to acellular yeast extract to ensure a constant glucose fermentation rate. The depletion of inorganic phosphate during in vitro fermentation led them to believe that it was incorporated into a sugar phosphate. They also observed that the yeast extract activity was due to a nondialyzable component, denaturable by heat, and a thermostable dialyzable component. They named these two components “zymase” and “cozymase.” Today, zymase is known to be a series of enzymes, and cozymase is composed of their cofactors as well as metal ions and ATP. The complete description of glycolysis dates back to the 1940s, due in particular to the contributions of Embden, Meyerhof, and Neuberg. For that reason, glycolysis is often called the Embden–Meyerhof–Parnas pathway.

The transport of must hexoses (glucose and fructose) across the plasma membrane activates a complex system of protein transporters that is not fully explained (Section 1.3.2). This mechanism facilitates the diffusion of must hexoses in the cytoplasm, where they are rapidly metabolized. Since solute moves in the direction of the concentration gradient, from the concentrated outer medium to the diluted inner medium, it is not an active transport system requiring energy. This is favorable from an energy standpoint.

Next, glycolysis (Figure 2.2) is carried out entirely in the cytosol of the cell. It includes a first stage, in which glucose is converted into fructose 1,6‐bisphosphate, requiring two ATP molecules. This transformation itself comprises three steps: an initial phosphorylation of glucose into glucose 6‐phosphate, the isomerization of the latter into fructose 6‐phosphate, and a second phosphorylation forming fructose 1,6‐bisphosphate. These three reactions are catalyzed by hexokinase, phosphoglucoisomerase, and phosphofructokinase, respectively.

In fact, Saccharomyces cerevisiae has two hexokinases (PI and PII) capable of phosphorylating glucose and fructose. Hexokinase PII is essential and is active predominantly during the yeast log phase in a sugar‐rich medium. Hexokinase PI, partially repressed by glucose, is not active until the stationary phase (Bisson, 1993).

Mutant strains devoid of phosphoglucoisomerase have been isolated. Their inability to develop on glucose suggests that glycolysis is the only catabolic pathway of glucose in S. cerevisiae (Caubet et al., 1988). The pentose phosphate pathway, by which some organisms utilize sugars, serves only as a means of synthesizing ribose 5‐phosphate, incorporated in nucleic acids and in reduced nicotinamide adenine dinucleotide phosphate (NADPH) in Saccharomyces.

The second stage of glycolysis forms glyceraldehyde 3‐phosphate. Under the catalytic action of aldolase, fructose 1,6‐bisphosphate is cleaved, thus forming two triose phosphate isomers: dihydroxyacetone phosphate and glyceraldehyde 3‐phosphate. Triose phosphate isomerase catalyzes the isomerization of these two compounds. Although at equilibrium, the ketose form is more abundant than the aldose form, the transformation of dihydroxyacetone phosphate into glyceraldehyde 3‐phosphate is rapid, since this compound is continually eliminated by the ensuing glycolysis reactions. In other words, a molecule of glucose leads to the formation of two molecules of glyceraldehyde 3‐phosphate.

The third phase of glycolysis is composed of two steps that recover part of the energy from glyceraldehyde 3‐phosphate. Initially, it is converted into 1,3‐bisphosphoglycerate (1,3‐BPG). This reaction is catalyzed by glyceraldehyde 3‐phosphate dehydrogenase. It is an oxidation coupled with a substrate‐level phosphorylation. Nicotinamide adenine dinucleotide (NAD⁺) is the 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.