Conversion of Six-Carbon Glucose toThree-Carbon Glyceraldehyde-3-Phosphate

Conversion of Six-Carbon Glucose toThree-Carbon Glyceraldehyde-3-Phosphate

The first steps of the glycolytic pathway prepare for the electron transfer and the eventual phosphorylation of ADP; these reactions make use of the free energy of hydrolysis of ATP. Figure 17.3 summarizes this part of the pathway, which is often called the preparation phase of glycolysis.

What reactions convert glucose-6-phosphate to glyceraldehyde-3-phosphate?

Step 1.Glucose is phosphorylated to give glucose-6-phosphate. Thephosphorylation of glucose is an endergonic reaction.

Glucose + P_i - > Glucose-6-phosphate + H₂O

∆G°' = 13.8 kJ mol^–1= 3.3 kcal mol^–1

The hydrolysis of ATP is exergonic.

ATP + H₂O - > ADP + P_i

∆G°' = –30.5 kJ mol^–1= –7.3 kcal mol^–1

These two reactions are coupled, so the overall reaction is the sum of the two and is exergonic.

Glucose + ATP - > Glucose-6-phosphate + ADP

∆G°' = (13.8 + –30.5) kJ mol^–1= –16.7 kJ mol^–1= –4.0 kcal mol^–1

Recall that ∆G°' is calculated under standard states with the concentration of all reactants and products at 1 M except hydrogen ion. If we look at the actual ΔΓGin the cell, the number varies depending on cell type and metabolic state,but a typical value for this reaction is –33.9 kJ mol^–1 or –8.12 kcal mol^–1. Thus the reaction is typically even more favorable under cellular conditions. Table 17.1 gives the ∆G°' and G values for all the reactions of anaerobic glycolysis in erythrocytes.

This reaction illustrates the use of chemical energy originally produced by the oxidation of nutrients and ultimately trapped by phosphorylation of ADP to ATP. Recall that ATP does not represent stored energy, just as an electric current does not represent stored energy. The chemical energy of nutrients is released by oxidation and is made available for immediate use on demand by being trapped as ATP.

The enzyme that catalyzes this reaction is hexokinase. The term kinase is applied to the class of ATP-dependent enzymes that transfer a phosphate group from ATP to a substrate. The substrate of hexokinase is not necessarily glucose; rather, it can be any one of a number of hexoses, such as glucose, fructose, and mannose. Glucose-6-phosphate inhibits the activity of hexokinase; this is a control point in the pathway. Some organisms or tissues contain multiple isozymes of hexokinase. One isoform of hexokinase found in the human liver, called glucokinase, lowers blood glucose levels after one has eaten a meal. Liver glucokinase requires a much higher substrate level to achieve saturation than hexokinase does. Because of this, when glucose levels are high, the liver can metabolize glucose via glycolysis preferentially over the other tissues. When glu-cose levels are low, hexokinase is still active in all tissues.

A large conformational change takes place in hexokinase when substrate is bound. It has been shown by X-ray crystallography that, in the absence of substrate, two lobes of the enzyme that surround the binding site are quite far apart. When glucose is bound, the two lobes move closer together, and the glu-cose becomes almost completely surrounded by protein (Figure 17.4).

This type of behavior is consistent with the induced-fit theory of enzyme action. In all kinases for which the structure is known, a cleft closes when substrate is bound.

Step 2.Glucose-6-phosphate isomerizes to give fructose-6-phosphate.Glucosephosphate isomerase is the enzyme that catalyzes this reaction. TheC-1 aldehyde group of glucose-6-phosphate is reduced to a hydroxyl group, and the C-2 hydroxyl group is oxidized to give the ketone group of fructose-6-phosphate, with no net oxidation or reduction. (Recall that glucose is an aldose, a sugar whose open-chain, noncyclic structure contains an aldehyde group, while fructose is a ketose, a sugar whose corresponding structure contains a ketone group.) The phosphorylated forms, glucose-6-phosphate and fructose-6-phosphate, are an aldose and a ketose, respectively.

Step 3.Fructose-6-phosphate is further phosphorylated, producing fructose- 1,6-bisphosphate.

As in the reaction in Step 1, the endergonic reaction of phosphorylation of fructose-6-phosphate is coupled to the exergonic reaction of hydrolysis of ATP, and the overall reaction is exergonic. See Table 17.1.

The reaction in which fructose-6-phosphate is phosphorylated to give fructose-1,6-bisphosphate is the one in which the sugar is committed to gly-colysis. Glucose-6-phosphate and fructose-6-phosphate can play roles in other pathways, but fructose-1,6-bisphosphate does not. After fructose-1,6-bisphos-phate is formed from the original sugar, no other pathways are available, and the molecule must undergo the rest of the reactions of glycolysis. The phos-phorylation of fructose-6-phosphate is highly exergonic and irreversible, and phosphofructokinase, the enzyme that catalyzes it, is the key regulatory enzymein glycolysis.

Phosphofructokinase is a tetramer that is subject to allosteric feedback regu-lation of the type we discussed. There are two types of subunits, designated M and L, that can combine into tetramers to give different per-mutations (M₄, M₃L, M₂L₂, ML₃, and L₄). These combinations of subunits are referred to as isozymes, and they have subtle physical and kinetic differences (Figure 17.5). The subunits differ slightly in amino acid composition, so the two isozymes can be separated from each other by electrophoresis. The tetrameric form that occurs in muscle is designated M₄, while that in liver is designated L₄. In red blood cells, several of the combinations can be found. Individuals who lack the gene that directs the synthesis of the M form of the enzyme can carry on glycolysis in their livers but experience muscle weakness because they lack the enzyme in muscle.

When the rate of the phosphofructokinase reaction is observed at varying concentrations of substrate (fructose-6-phosphate), the sigmoidal curve typical of allosteric enzymes is obtained. ATP is an allosteric effector in the reaction. High levels of ATP depress the rate of the reaction, and low levels of ATP stimulate the reaction (Figure 17.6). When there is a high level of ATP in the cell, a good deal of chemical energy is immediately available from hydrolysis of ATP. The cell does not need to metabolize glucose for energy, so the presence of ATP inhibits the glycolytic pathway at this point. There is also another, more potent, allosteric effector of phosphofructokinase. This effector is fructose-2,6-bisphosphate; we shall discuss its mode of action when we consider general control mechanisms in carbohydrate metabolism.

Step 4.Fructose-1,6-bisphosphate is split into two three-carbon fragments. Thecleavage reaction here is the reverse of an aldol condensation; the enzyme that catalyzes it is called aldolase. In the enzyme isolated from most animal sources (the one from muscle is the most extensively studied), the basic side chain of an essential lysine residue plays the key role in catalyzing this reaction. The thiol group of a cysteine also acts as a base here.

Step 5. The dihydroxyacetone phosphate is converted to glyceraldehyde-3- phosphate.

The enzyme that catalyzes this reaction is triosephosphate isomerase. (Both dihydroxyacetone and glyceraldehyde are trioses.)

One molecule of glyceraldehyde-3-phosphate has already been produced by the aldolase reaction; we now have a second molecule of glyceraldehyde-3-phosphate, produced by the triosephosphate isomerase reaction. The original molecule of glucose, which contains six carbon atoms, has now been converted to two molecules of glyceraldehyde-3-phosphate, each of which contains three carbon atoms.

The ∆G value for this reaction under physiological conditions is slightly positive (+2.41 kJ mol^–1 or +0.58 kcal mol^–1). It might be tempting to think that the reaction would not occur and that glycolysis would be halted at this step. We must remember that just as coupled reactions involving ATP hydrolysis add their G values together for the overall reaction, glycolysis is composed of many reactions that have very negative G values that can drive the reaction to completion. A few reactions in glycolysis have small, positive ∆G values (see Table 17.1), but four reactions have very large, negative values, so that the ∆G for the whole process is negative.

Summary

In the first stages of glycolysis, glucose is converted to two molecules of glyceraldehyde-3-phosphate.

The key intermediate in this series of reactions is fructose-1,6-bisphosphate. The reaction that produces this intermediate is a keycontrol point of the pathway, and the enzyme that catalyzes it, phospho-fructokinase, is subject to allosteric regulation.