Glycolysis: From Glucose to Pyruvate

We begin our discussion of the energy-harvesting pathways with glycolysis which begins glucose metabolism. Glycolysis takes place in the cytoplasm of cells. It converts glucose to pyruvate, produces a small amount of energy and does not generate CO₂. In glycolysis, a reduced fuel molecule, glucose, gets partially oxidized and in the process releases some of its energy. After ten enzyme-catalyzed reactions, the end products of glycolysis are two molecules of pyruvate (pyruvic acid)*. These reactions are accompanied by the net formation of two molecules of ATP and by the reduction of two molecules of NAD+ to two molecules of NADH + H+ for each molecule of glucose. Glycolysis can be divided into two stages: energy-investing reactions that use ATP, and energy-harvesting reactions that produce ATP.

The energy-investing reactions of glycolysis require ATP

The first five reactions of glycolysis are endergonic; that is, the cell is investing free energy in the glucose molecule, rather than releasing energy from it. In two separate reactions, the energy of two molecules of ATP is invested in attaching two phosphate groups to the glucose molecule to form fructose 1,6-bisphosphate,* which has a free energy substantially higher than that of glucose. Later, these phosphate groups will be transferred to ADP to make new molecules of ATP. Although both of these first steps of glycolysis use ATP as one of their substrates, each is catalyzed by a different, specific enzyme. The enzyme hexokinase catalyzes reaction 1, in which a phosphate group from ATP is attached to the sixcarbon glucose molecule, forming glucose 6-phosphate. (A kinase is any enzyme that catalyzes the transfer of a phosphate group from ATP to another substrate.) In reaction 2, the six-membered glucose ring is rearranged into a fivemembered fructose ring. In reaction 3, the enzyme phosphofructokinase adds a second phosphate (taken from another ATP) to the fructose ring, forming a six-carbon sugar, fructose 1,6-bisphosphate. Reaction 4opens up and cleaves the six-carbon sugar ring to give two different three-carbon sugar phosphates: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. In reaction 5, one of those products, dihydroxyacetone phosphate, is converted into a second molecule of the other one, glyceraldehyde 3-phosphate (G3P). By this time—the halfway point of the glycolytic pathway— the following things have happened:

Two molecules of ATP have been invested.

The six-carbon glucose molecule has been converted into two molecules of a three-carbon sugar phosphate, glyceraldehydes 3-phosphate (G3P, a triose phosphate).

The energy-harvesting reactions of glycolysis yield NADH + H+ and ATP

With the investment of two ATPs, the first five reactions of glycolysis have rearranged the six-carbon sugar glucose and split it into two three-carbon sugar phosphates (G3P). In the discussion that follows, remember that each reaction occurs twice for each glucose molecule going through glycolysis because each glucose molecule has been split into two molecules of G3P. It is the fate of G3P that now concerns us—its transformation will generate both NADH + H+ and ATP.

Producing NADH + H+.

Reaction 6is catalyzed by the enzyme triose phosphate dehydrogenase, and its end product is a phosphate ester, 1,3-bisphosphoglycerate (BPG). Reaction 6 is an oxidation and it is accompanied by an enormous drop in free energy — more than 100 kcal of energy per mole of glucose is released in this extremely exergonic reaction. If this big energy drop were simply a loss of heat, glycolysis would not provide useful energy to the cell. However, rather than being lost as heat, this energy is stored as chemical energy by reducing two molecules of NAD+ to make two molecules of NADH + H+. Because NAD+ is present in small amounts in the cell, it must be recycled to allow glycolysis to continue; if none of the NADH is oxidized back to NAD+, glycolysis comes to a halt. The metabolic pathways that follow glycolysis carry out this oxidation, as we will see.

Producing ATP.

In reactions 7–10, the two phosphate groups of BPG are transferred one at a time to molecules of ADP, with a rearrangement in between. More than 20 kcal (83.6 kJ/mol) of free energy is stored in ATP for every mole of BPG broken down. Finally, we are left with two moles of pyruvate for every mole of glucose that entered glycolysis.

The enzyme-catalyzed transfer of phosphate groups from donor molecules to ADP molecules (as in reaction 7) is called substrate-level phosphorylation. (Phosphorylation is the addition of a phosphate group to a molecule. Substrate-level phosphorylation is distinguished from the oxidative phosphorylation carried out by the respiratory chain, which we will discuss later in the chapter.) As an example of substrate-level phosphorylation, when G3P reacts with a phosphate group (Pi) and NAD+ in reaction 6, a second phosphate is added, an aldehyde is oxidized to a carboxylic acid, NAD+ is reduced and BPG is formed. The oxidation provides so much energy that the newly added phosphate group is linked to the rest of the molecule by a covalent bond that has even more energy than the terminal phosphate-to-phosphate bond of ATP. Another example of substrate-level phosphorylation occurs in reaction 7, where phosphoglycerate kinase catalyzes the transfer of a phosphate group from BPG to ADP forming ATP. Both reactions 6 and 7 are exergonic, even though a substantial amount of energy is consumed in the formation of ATP. A review of the glycolytic pathway shows that at the beginning of glycolysis, two molecules of ATP are used per molecule of glucose, but that ultimately four molecules of ATP are produced (two for each of the two BPG molecules)— a net gain of two ATP molecules and two NADH + H+.

Glycolysis is followed by cellular respiration (if O₂ is present) or fermentation (if no O₂ is present). The first reaction of cellular respiration is the oxidation of pyruvate.

Pyruvate Oxidation

The oxidation of pyruvate to acetate and its subsequent conversion to acetyl CoAis the link between glycolysis and all the other reactions of cellular respiration.

Coenzyme A(CoA), which is attached to the acetyl group to form acetyl CoA, is a complex molecule composed of a nucleotide, the vitamin pantothenic acid and a sulfur-containing group that is responsible for binding the two-carbon acetate molecule. Acetyl CoA formation is a multi-step reaction catalyzed by the pyruvate dehydrogenase complex, an enormous enzyme complex that is attached to the inner mitochondrial membrane. Pyruvate diffuses into the mitochondrion where a series of coupled reactions takes place:

1. Pyruvate is oxidized to a two-carbon acetyl group, and CO₂ is released.

2. Part of the energy from the oxidation is captured by the reduction of NAD+ to NADH + H+.

3. Some of the remaining energy is stored temporarily by the combining of the acetyl group with CoA, forming acetyl CoA: pyruvate + NAD+ + CoA®Acetyl CoA + NADH + H+ + CO₂ Acetyl CoA has 7.5 kcal/mol (31.4 kJ/mol) more energy than simple acetate. Acetyl CoA can donate the acetyl group to acceptor molecules, much as ATP can donate phosphate groups to various acceptors. In the next section, we will see that the acetyl CoA donates its acetyl group to the four-carbon compound oxaloacetate to form the six-carbon citrate.