6.3: Some Details of Glycolysis

Here we focus on the enzyme-catalyzed reactions and free energy transfers between pathway components, looking at the energetics and enzymatic features of each reaction.

6.3.1. Glycolysis, Stage 1

Reaction 1: In the first reaction of glycolysis, hexokinase rapidly phosphorylates glucose entering the cell, forming glucose-6-phosphate (G-6-P). Figure 6.6 (below) shows that the overall reaction is exergonic. The standard free energy change for the reaction is −4 Kcal per mole of G-6-P synthesized.

Figure 6.6: In reaction 1, phosphorylation of glucose to make glucose-6-phosphate (G-6-P) consumes a molecule of ATP. If cellular energy needs are being met, G-6-P will be polymerized to make storage polysaccharides. In animals, G-6-P can be retrieved by glycogen hydrolysis to G-1-P by glycogen phosphorylase when the cells require nutrient energy; at that time, the G-6-P will resume glycolysis.

The hexokinase reaction is a coupled reaction, in which phosphorylation of glucose is coupled to ATP hydrolysis. The free energy of ATP hydrolysis (an energetically favorable reaction) fuels glucose phosphorylation (an energetically unfavorable reaction). The reaction is also biologically irreversible, as shown by the single vertical arrow.

Excess dietary glucose can be stored in most cells (especially liver and kidney cells) as glycogen—a highly branched polymer of glucose monomers. In green algae and plants, glucose made by photosynthesis is stored as polymers of starch. When glucose is necessary for energy, glycogen and starch hydrolysis form glucose-1-phosphate (G-1-P), which is then converted to G-6-P. Let’s look at the energetics, i.e., the flow of free energy in the hexokinase-catalyzed reaction. This reaction can be seen as the sum of two reactions in Figure 6.7

Figure 6.7: Free energy flow (exchange) for reaction 1 of glycolysis, and for the removal (hydrolysis) of the phosphate from G-6-P. Can you figure out the standard free energy change for the hydrolysis of G-6-P?

Recall that ATP hydrolysis is an exergonic reaction, releasing about 7 (~7) Kcal/mol (rounding down!) in a closed system under standard conditions. The condensation reaction of glucose phosphorylation occurs with a \(\Delta Go\) of +3 Kcal/mol. Under standard conditions, this is an endergonic reaction. Summing up the free energy changes of the two reactions, we can calculate the overall \(\Delta Go\) of −4 Kcal/mol for the coupled reaction under standard conditions in a closed system.

The reactions in Figure 6.7 are written as if they are reversible. However, we said that the overall coupled reaction is biologically irreversible. Why the contradiction? To explain, we say that an enzyme-catalyzed reaction is biologically irreversible when the products have a relatively low affinity for the enzyme’s active site, making catalysis (acceleration) of the reverse reaction very inefficient. While enzymes catalyzing biologically irreversible reactions don’t facilitate the return of products back into reactants, they are often allosterically regulated. This is the case for hexokinase. Imagine a cell that slows its consumption of G-6-P because its energy needs are being met. As a result, G-6-P levels rise in cells. As you might expect, the hexokinase reaction slows down so that the cell doesn’t unnecessarily consume a precious nutrient free energy resource. The allosteric regulation of hexokinase is shown in Figure 6.8.

Figure 6.8: Enzymatics of the hexokinase reaction: this enzyme catalyzes a biologically irreversible reaction and is allosterically regulated.

As G-6-P concentrations rise in the cell, excess G-6-P binds to an allosteric site on hexokinase. The resulting conformational change in the enzyme is then transferred to the active site, inhibiting the glucose phosphorylation reaction. The inhibition is reversible: when G-6-P levels decline in the cell, it comes off of the enzyme, the allosteric change is reversed and uninhibited reaction rates resume.

152-2 Glycolysis Stage 1, Reaction 1

CHALLENGE

Hexokinase regulation was selected in evolution. Speculate on the benefit of this trait.

Reaction 2: In this slightly endergonic and reversible reaction, isomerase catalyzes the isomerization of G-6-P to fructose-6-P (F-6-P). The reaction is shown below in Figure 6.9.

Figure 6.9: Reaction 2 of glycolysis is the isomerization of G-6-P to F-6-P by an isomerase (fructose is a structural isomer of glucose). The reaction is endergonic and reversible.

Reaction 3: In this biologically irreversible reaction, 6-P-fructokinase catalyzes the phosphorylation of F-6-P to make fructose 1,6-diphosphate (F1,6-diP). In this coupled reaction, ATP again provides the second phosphate. The overall reaction is written as the sum of two reactions in Figure 6.10.

Figure 6.10: In reaction 3 of glycolysis, a kinase catalyzes the phosphorylation of F-6-P to F1,6-diP in a biologically irreversible reaction, consuming molecule of ATP.

Like the hexokinase reaction, the 6-P-fructokinase reaction is a coupled, exergonic, and allosterically regulated reaction. Multiple allosteric effectors, including ATP, ADP, AMP, and long-chain fatty acids, regulate this enzyme.

CHALLENGE

Explain when and how each of the different allosteric effectors regulates this kinase-catalyzed reaction.

Reactions 4 and 5: These are the last reactions of the first stage of glycolysis. In reaction 4, F1,6-diP (a 6-C sugar) is reversibly split into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P). In reaction 5 (also reversible), DHAP is converted into another G-3-P. These reactions are shown in Figure 6.11.

Figure 6.11: In reaction 4, F1,6-diP is split into glyceraldehyde-3-P (G-3-P) and dihydroxyacetone phosphate (DHAP) by an aldolase. In reaction 5, DHAP is isomerized to G-3-P. Both reactions are endergonic, consuming free energy. The two reactions mark the end of Stage 1 of glycolysis.

The net result is the formation of two molecules of G-3-P at the end of the reactions of Stage 1. The enzymes F-diP aldolase and triose-P-isomerase both catalyze freely reversible reactions. Also, both reactions proceed with a positive free energy change and are therefore endergonic. The sum of the free energy changes for splitting F1,6-diP into two G-3-Ps is a whopping +7.5 Kcal/mol, a very energetically unfavorable process.

CHALLENGE

Given that these reactions are both endergonic, how is it possible that glycolysis ever gets past the synthesis of F1,6-diP?

In summary, by the end of Stage 1 of glycolysis, two ATP molecules have been consumed, and one 6-C carbohydrate has been split into two 3-C carbohydrates. We have also seen two biologically irreversible and allosterically regulated enzymes.

153 Glycolysis Stage 1, Reactions 2-5

6.3.2. Glycolysis, Stage 2

We will follow just one of the two molecules of G-3-P generated by the end of Stage 1 of glycolysis but remember that both proceed through Stage 2 (the remainder) of glycolysis.

Reaction 6: In this redox reaction, G-3-P is oxidized to 1,3-diphosphoglyceric acid (1,3- diPG), and NAD+ is reduced to NADH. The reaction catalyzed by the G-3-P dehydrogenase enzyme, as shown in Figure 6.12.

Figure 6.12: Reaction 6, a redox reaction catalyzed by G-3-P dehydrogenase, is freely reversible. hydride (\(\rm H^{-}\)) ions are transferred to \(\rm NAD^{+}\) from G-3-P to make NADH and 1,3 diphosphoglyceric acid (1,3diPG).

This freely reversible endergonic redox reaction, removes a hydrogen molecule (\(H_2\) from G-3-P, leaving behind phosphoglyceric acid. This short-lived oxidation intermediate is phosphorylated to make 1,3 diphosphoglyceric acid (1,3diPG). At the same time, the hydrogen molecule is split into a hydride ion (\(\rm H^{-}\)) and a proton (\(\rm H^{+}\)). The \(\rm H^{-}\) ions reduce \(\rm NAD^{+}\) to NADH, leaving the protons behind in solution. Remember that all of this is happening in the active site of the same enzyme! Remind yourself of what is oxidized and what is reduced here!

Even though it catalyzes a reversible reaction, G-3-P dehydrogenase is allosterically regulated. However, in contrast to the regulation of hexokinase, that of G-3-P dehydrogenase is more complicated! The regulator is \(\rm NAD^{+}\), and the mechanism of allosteric regulation is called negative cooperativity. It turns out that the higher the [\(\rm NAD^{+}\)] in the cell, the lower the affinity of the enzyme for more \(\rm NAD^{+}\) and the faster the reaction in the cell! The mechanism is discussed at the following link.

154 Glycolysis Stage 2, Reaction 6

CHALLENGE

How might you explain the logic of negative cooperativity in regulating 1,3-diPG formation?

Reaction 7: This reaction, catalyzed by phosphoglycerate kinase, is freely reversible and exergonic (Figure 6.13), yielding ATP and 3-phosphoglyceric acid (3PG).

Figure 6.13: Reaction 7—a reversible, exergonic, coupled reaction generates ATP using free energy released by the hydrolysis of one of the phosphates from 1,3diPG. The mechanism of ATP synthesis here is called substrate-level phosphorylation.

In glycolysis, catalysis of phosphate group transfer between molecules by kinases is called substrate-level phosphorylation, one of the ways of phosphorylating ADP to make ATP. In this coupled reaction, the free energy released by hydrolyzing a phosphate from 1,3diPG is used to make ATP.

Remember that this reaction occurs twice per starting molecule of glucose, so that two ATPs have been synthesized during this reaction of glycolysis. We call 1,3diPG a very high energy phosphate compound.

Reaction 8: This freely reversible endergonic reaction transfers the phosphate from the number 3-C of 3PG to the number 2-C (Figure 6.14). Mutases like phosphoglyceromutase catalyze the transfer of functional groups within a molecule.

Figure 6.14: Reaction 8 is a reversible, endergonic reaction catalyzed by a mutase. Mutases catalyze the transfer of a chemical group from one part of a molecule to another. Here, 3-PG is converted to 2-PG.

Reaction 9: In this reaction (Figure 6.15), enolase catalyzes the conversion of 2-PG to phosphoenolpyruvate (PEP).

Figure 6.15: Reaction 9 is a reversible endergonic reaction. An enolase catalyzes the conversion of 2-PG to phosphoenolpyruvate (PEP).

Reaction 10: This reaction results in the formation of a molecule of pyruvic acid (pyruvate), illustrated in Figure 6.16.

Figure 6.16: Reaction 10 is biologically irreversible. In a coupled reaction, pyruvate kinase catalyzes a highly exergonic transfer of a phosphate on PEP to ADP to make ATP.

Remember again that two pyruvates are produced per starting glucose molecule. The enzyme pyruvate kinase couples the biologically irreversible, exergonic hydrolysis of a phosphate from PEP and the transfer of that phosphate to ADP in a coupled reaction. The reaction produces PEP, another very high-energy phosphate compound.

155-2 Glycolysis Stage 2, Reactions 7-10

CHALLENGE

1,3-diPG and PEP are very high-energy phosphate compounds compared to ATP, which is just called a high-energy-phosphate compound. Explain the difference.

Pyruvate kinase is allosterically regulated by increases in cellular levels of ATP, citric acid, long-chain fatty acids, F1,6-diP—or even PEP, one of its own substrates.

CHALLENGE

Predict and explain how each of these allosteric effectors should change the rate of pyruvate kinase catalysis.

As we have seen, there are alternate fates of pyruvate, the product of incomplete glycolysis. One is the aerobic mitochondrial oxidation of pyruvate, following incomplete glycolysis. The other is an anaerobic fermentation, or complete glycolysis, in which pyruvate is reduced to one or another end product. Recall that muscle fatigue results when skeletal muscle uses anaerobic fermentation to get the free energy required for vigorous exercise by reducing pyruvate to lactic acid. It is the accumulation of lactic acid in skeletal-muscle cells that causes muscle fatigue. The enzyme LDH (lactate dehydrogenase) that catalyzes the reduction of pyruvate to lactate is regulated—but not allosterically! Instead, different types of muscle tissues regulate LDH by making different versions of the enzyme. Go to the following link for a more detailed explanation.

156 Fermentation: Regulation of Pyruvate Regulation Is NOT Allosteric!