glycolysis · from the Greek glyk-, sweet, and lysis, splitting

Nearly every living cell carries out a catabolic process known as glycolysis—the stepwise degradation of glucose (and other simple sugars). Glycolysis is a paradigm of metabolic pathways. Carried out in the cytosol of cells, it is basically an anaerobic process; its principal steps occur with no requirement for oxygen. Living things first appeared in an environment lacking O₂, and glycolysis was an early and important pathway for extracting energy from nutrient molecules. It played a central role in anaerobic metabolic processes during the first 2 billion years of biological evolution on earth. Modern organisms still employ glycolysis to provide precursor molecules for aerobic catabolic pathways (such as the tricarboxylic acid cycle) and as a short-term energy source when oxygen is limiting.

19.1 · Overview of Glycolysis

An overview of the glycolytic pathway is presented in Figure 19.1. Most of the details of this pathway (the first metabolic pathway to be elucidated) were worked out in the first half of the 20th century by the German biochemists Otto Warburg, G. Embden, and O. Meyerhof. In fact, the sequence of reactions in Figure 19.1 is often referred to as the Embden-Meyerhof pathway.

Figure 19.1• The glycolytic pathway.

                Glycolysis consists of two phases. In the first, a series of five reactions, glucose is broken down to two molecules of glyceraldehyde-3-phosphate. In the second phase, five subsequent reactions convert these two molecules of glyceraldehyde-3-phosphate into two molecules of pyruvate. Phase 1 consumes two molecules of ATP (Figure 19.2). The later stages of glycolysis result in the production of four molecules of ATP. The net is 4 – 2 = 2 molecules of ATP produced per molecule of glucose.

Figure 19.2 • In the first phase of glycolysis, five reactions convert a molecule of glucose to two molecules of glyceraldehyde-3-phosphate.

Rates and Regulation of Glycolytic Reactions Vary Among Species

Microorganisms, plants, and animals (including humans) carry out the 10 reactions of glycolysis in more or less similar fashion, although the rates of the individual reactions and the means by which they are regulated differ from species to species. The most significant difference among species, however, is the way in which the product pyruvate is utilized. The three possible paths for pyruvate are shown in Figure 19.1. In aerobic organisms, including humans, pyruvate is oxidized (with loss of the carboxyl group as CO₂), and the remaining two-carbon unit becomes the acetyl group of acetyl-coenzyme A. This acetyl group is metabolized by the tricarboxylic acid cycle (and fully oxidized) to yield CO₂. The electrons removed in this oxidation process are subsequently passed through the mitochondrial electron transport system and used to generate molecules of ATP by oxidative phosphorylation, thus capturing most of the metabolic energy available in the original glucose molecule.

19.2  ·   The Importance of Coupled Reactions in Glycolysis

The process of glycolysis converts some, but not all, of the metabolic energy of the glucose molecule into ATP. The free energy change for the conversion of glucose to two molecules of lactate (the anaerobic route shown in Figure 19.1) is -183.6 kJ/mol:

C₆H₁₂0₆ ® 2H₃C—CHOH—COO^- + 2H⁺                            
DG°' = 2183.6 kJ/mol                    (19.1)

This process occurs with no net oxidation or reduction. Although several individual steps in the pathway involve oxidation or reduction, these steps compensate each other exactly. Thus, the conversion of a molecule of glucose to two molecules of lactate involves simply a rearrangement of bonds, with no net loss or gain of electrons. The energy made available through this rearrangement into a more stable (lower energy) form is a relatively small part of the total energy obtainable from glucose.

                The production of two molecules of ATP in glycolysis is an energy-requiring process:

2ADP |+ 2P_i ® 2ATP + 2H₂O         
DG°' = 2 x 30.5kJ/mol = 61.0 kJ/mol    (19.2)

Glycolysis couples these two reactions:

Glucose |+ 2ADP + 2P_i ® 2 lactate + 2ATP + 2H⁺ + 2H₂O                              
DG°' = 2183.6 + 61 = 2122.6 kJ/mol                    (19.3)

Thus, under standard-state conditions, (61/183.6) x 100%, or 33%, of the free energy released is preserved in the form of ATP in these reactions. However, as we discussed in Chapter 3, the various solution conditions, such as pH, concentration, ionic strength, and presence of metal ions, can substantially alter the free energy change for such reactions. Under actual cellular conditions, the free energy change for the synthesis of ATP (Equation 19.2) is much larger, and approximately 50% of the available free energy is converted into ATP. Clearly, then, more than enough free energy is available in the conversion of glucose into lactate to drive the synthesis of two molecules of ATP.

19.3  · The First Phase of Glycolysis

One way to synthesize ATP using the metabolic free energy contained in the glucose molecule would be to convert glucose into one (or more) of the high-energy phosphates in Table 3.3 that have standard-state free energies of hydrol ysis more negative than that of ATP. Those molecules in Table 3.3 that can be synthesized easily from glucose are phosphoenolpyruvate, 1,3-bisphosphoglycerate, and acetyl phosphate. In fact, in the first stage of glycolysis, glucose is converted into two molecules of glyceraldehyde-3-phosphate. Energy released from this high-energy molecule in the second phase of glycolysis is then used to synthesize ATP.

Reaction 1: Phosphorylation of Glucose by Hexokinase or Glucokinase —The First Priming Reaction

The initial reaction of the glycolysis pathway involves phosphorylation of glucose at carbon atom 6 by either hexokinase or glucokinase. The formation of such a phosphoester is thermodynamically unfavorable and requires energy input to operate in the forward direction (Chapter 3). The energy comes from ATP, a requirement that at first seems counterproductive. Glycolysis is designed to make ATP, not consume it. However, the hexokinase, glucokinase reaction (Figure 19.2) is one of two priming reactions in the cycle. Just as old-fashioned, hand-operated water pumps (Figure 19.3) have to be primed with a small amount of water to deliver more water to the thirsty pumper, the glycolysis pathway requires two priming ATP molecules to start the sequence of reactions and delivers four molecules of ATP in the end.

Figure 19.3 • Just as a water pump must be “primed” with water to get more water out, the glycolytic pathway is primed with ATP in steps 1 and 3 in order to achieve net production of ATP in the second phase of the pathway. (Michelle Sassi/The Stock Market)

                The complete reaction for the first step in glycolysis is

a-D-Glucose |+ ADP^4- ® a-D-glucose-6-phosphate^2- + ADP^3- + H⁺                                               
                                                          DG°' = 216.7 kJ/mol                                           (19.4)               

The hydrolysis of ATP makes 30.5 kJ/mol available in this reaction, and the phosphorylation of glucose “costs” 13.8 kJ/mol (see Table 19.1). Thus, the reaction liberates 16.7 kJ/mol under standard-state conditions (1M concentrations), and the equilibrium of the reaction lies far to the right ( K_eq = 850 at 25°C; see Table 19.1).

Table 19.1
Reactions and Thermodynamics of Glycolysis
Reaction	Enzyme	Source	Subunit Molecular Weight ( Mr )	Oligomeric Composition	DG°' (kJ/mol)	Keq at 25°C	DG (kJ/mol)
	Hexokinase	Mammals	100,000	Monomer	-16.7	850	-33.9*
	Hexokinase	Yeast	55,000	Dimer
	Glucokinase	Mammalian liver	50,000	Monomer
	Phosphoglucoisomerase	Human	65,000	Dimer	+1.67	0.51	-2.92
	Phosphofructokinase	Rabbit muscle	78,000	Tetramer	-14.2	310	-18.8
	Fructose bisphosphate aldolase	Rabbit muscle	40,000	Tetramer	+23.9	6.43 x 10-5	-0.23
	Triose phosphate isomerase	Chicken muscle	27,000	Dimer	+7.56	0.0472	+2.41
	Glyceraldehyde-3-P dehydrogenase	Rabbit muscle	37,000	Tetramer	+6.30	0.0786	-1.29
	Phosphoglycerate kinase	Rabbit muscle	64,000	Monomer	-18.9	2060	+0.1
	Phosphoglycerate mutase	Rabbit muscle	27,000	Dimer	+4.4	0.169	+0.83
	Enolase	Rabbit muscle	41,000	Dimer	+1.8	0.483	+1.1
	Pyruvate kinase	Rabbit muscle	57,000	Tetramer	-31.7	3.63 x 105	-23.0
	Lactate dehydrogenase	Rabbit muscle	35,000	Tetramer	-25.2	2.63 x 104	-14.8
* DG values calculated for 310 K (37°C) using the data in Table 19.2 for metabolite concentrations in erythrocytes. DG°' values are assumed to be the same at 25°C and 37°C.

                Under cellular conditions, this first reaction of glycolysis is even more favorable than at standard state. As pointed out in Chapter 3, the free energy change for any reaction depends on the concentrations of reactants and products. Equation 3.12 in Chapter 3 and the data in Table 19.2 can be used to calculate a value for DG for the hexokinase, glucokinase reaction in erythrocytes:

Table 19.2

         (19.5)

Thus, DG is even more favorable under cellular conditions than at standard state. As we will see later in this chapter, the hexokinase, glucokinase reaction is one of several that drive glycolysis forward.

The Cellular Advantages of Phosphorylating Glucose

The incorporation of a phosphate into glucose in this energetically favorable reaction is important for several reasons. First, phosphorylation keeps the substrate in the cell. Glucose is a neutral molecule and could diffuse across the cell membrane, but phosphorylation confers a negative charge on glucose, and the plasma membrane is essentially impermeable to glucose-6-phosphate (Figure 19.4). Moreover, rapid conversion of glucose to glucose-6-phosphate keeps the intracellular concentration of glucose low, favoring diffusion of glucose into the cell. In addition, because regulatory control can be imposed only on reactions not at equilibrium, the favorable thermodynamics of this first reaction makes it an important site for regulation.

Figure 19.4 • Phosphorylation of glucose to glucose-6-phosphate by ATP creates a charged molecule that cannot easily cross the plasma membrane.

Hexokinase

In most animal, plant, and microbial cells, the enzyme that phosphorylates glucose is hexokinase. Magnesium ion (Mg²⁺) is required for this reaction, as for the other kinase enzymes in the glycolytic pathway. The true substrate for the hexokinase reaction is MgATP^2-. The apparent K_m for glucose of the animal skeletal muscle enzyme is approximately 0.1 mM, and the enzyme thus operates efficiently at normal blood glucose levels of 4 mM or so. Different body tissues possess different isozymes of hexokinase, each exhibiting somewhat different kinetic properties. The animal enzyme is allosterically inhibited by the product, glucose-6-phosphate. High levels of glucose-6-phosphate inhibit hexokinase activity until consumption by glycolysis lowers its concentration. The hexokinase reaction is one of three points in the glycolysis pathway that are regulated. As the generic name implies, hexokinase can phosphorylate a variety of hexose sugars, including glucose, mannose, and fructose.

Glucokinase

Liver contains an enzyme called glucokinase , which also carries out the reaction in Figure 19.4 but is highly specific for _D-glucose, has a much higher K_m for glucose (approximately 10.0 mM ), and is not product-inhibited. With such a high K_m for glucose, glucokinase becomes important metabolically only when liver glucose levels are high (for example, when the individual has consumed large amounts of sugar). When glucose levels are low, hexokinase is primarily responsible for phosphorylating glucose. However, when glucose levels are high, glucose is converted by glucokinase to glucose-6-phosphate and is eventually stored in the liver as glycogen. Glucokinase is an inducible enzyme—the amount present in the liver is controlled by insulin (secreted by the pancreas). (Patients with diabetes mellitus produce insufficient insulin. They have low levels of glucokinase, cannot tolerate high levels of blood glucose, and produce little liver glycogen.) Because glucose-6-phosphate is common to several metabolic pathways (Figure 19.5), it occupies a branch point in glucose metabolism.

Figure 19.5 • Glucose-6-phosphate is the branch point for several metabolic pathways.

Reaction 2: Phosphoglucoisomerase Catalyzes the Isomerization of Glucose-6-Phosphate

The second step in glycolysis is a common type of metabolic reaction: the isom-erization of a sugar. In this particular case, the carbonyl oxygen of glucose-6-phosphate is shifted from C-1 to C-2. This amounts to isomerization of an aldose (glucose-6-phosphate) to a ketose—fructose-6-phosphate (Figure 19.6). The reaction is necessary for two reasons. First, the next step in glycolysis is phosphorylation at C-1, and the hemiacetal -OH of glucose would be more difficult to phosphorylate than a simple primary hydroxyl. Second, the isomerization to fructose (with a carbonyl group at position 2 in the linear form) activates carbon C-3 for cleavage in the fourth step of glycolysis. The enzyme responsible for this isomerization is phosphoglucoisomerase , also known as glucose phosphate isomerase. In humans, the enzyme requires Mg²⁺ for activity and is highly specific for glucose-6-phosphate. The DG°' is 1.67 kJ/mol, and the value of DG under cellular conditions (Table 19.1) is -2.92 kJ/mol. This small value means that the reaction operates near equilibrium in the cell and is readily reversible. Phosphoglucoisomerase proceeds through an enediol intermediate, as shown in Figure 19.6. Although the predominant forms of glucose-6-phosphate and fructose-6-phosphate in solution are the ring forms (Figure 19.6), the isomerase interconverts the open-chain form of G-6-P with the open-chain form of F-6-P. The first reaction catalyzed by the isomerase is the opening of the pyranose ring (Figure 19.6, Step A). In the next step, the C-2 proton is removed from the substrate by a basic residue on the enzyme, facilitating formation of the enediol intermediate (Figure 19.6, Step B). This process then operates somewhat in reverse (Figure 19.6, Step C), creating a carbonyl group at C-2 to complete the formation of fructose-6-phosphate. The furanose form of the product is formed in the usual manner by attack of the C-5 hydroxyl on the carbonyl group, as shown.

Figure 19.6 • The phosphoglucoisomerase mechanism involves opening of the pyranose ring (Step A), proton abstraction leading to enediol formation (Step B), and proton addition to the double bond, followed by ring closure (Step C).

Reaction 3: Phosphofructokinase —The Second Priming Reaction

The action of phosphoglucoisomerase, “moving” the carbonyl group from C-1 to C-2, creates a new primary alcohol function at C-1 (see Figure 19.5). The next step in the glycolytic pathway is the phosphorylation of this group by phosphofructokinase. Once again, the substrate that provides the phosphoryl group is ATP. Like the hexokinase, glucokinase reaction, the phosphorylation of fructose-6-phosphate is a priming reaction and is endergonic :

Fructose-6-P + P_i ® fructose-1,6-biophosphate                                                
                                                           DG°' = 16.3 kJ/mol                                            (19.6) 

When coupled (by phosphofructokinase) with the hydrolysis of ATP, the overall reaction (Figure 19.7) is strongly exergonic:

Figure 19.7 • The phosphofructokinase reaction.

Fructose-6-P + ATP ® fructose-1,6-biophosphate +ADP
                           DG°' = 214.2 kJ/mol                                                  (19.7)
                                                       DG (in erythrocytes) = 218.8 kJ/mol                                                                
                                                                    

At pH 7 and 37°C, the phosphofructokinase reaction equilibrium lies far to the right. Just as the hexokinase reaction commits the cell to taking up glucose, the phosphofructokinase reaction commits the cell to metabolizing glucose rather than converting it to another sugar or storing it. Similarly, just as the large free energy change of the hexokinase reaction makes it a likely candidate for regulation, so the phosphofructokinase reaction is an important site of regulation—indeed, the most important site in the glycolytic pathway.

Regulation of Phosphofructokinase

Phosphofructokinase is the “valve” controlling the rate of glycolysis. ATP is an allosteric inhibitor of this enzyme. In the presence of high ATP concentrations, phosphofructokinase behaves cooperatively, plots of enzyme activity versus ­fructose-6-phosphate are sigmoid, and the K_m for fructose-6-phosphate is increased (Figure 19.8). Thus, when ATP levels are sufficiently high in the cytosol, glycolysis “turns off.” Under most cellular conditions, however, the ATP concentration does not vary over a large range. The ATP concentration in muscle during vigorous exercise, for example, is only about 10% lower than that during the resting state. The rate of glycolysis, however, varies much more. A large range of glycolytic rates cannot be directly accounted for by only a 10% change in ATP levels.

Figure 19.8 • At high [ATP], phosphofructokinase (PFK) behaves cooperatively, and the plot of enzyme activity versus [fructose-6-phosphate] is sigmoid. High [ATP] thus inhibits PFK, decreasing the enzyme’s affinity for fructose-6-phosphate.

                AMP reverses the inhibition due to ATP, and AMP levels in cells can rise dramatically when ATP levels decrease, due to the action of the enzyme adenylate kinase, which catalyzes the reaction

with the equilibrium constant:

(19.8)

Adenylate kinase rapidly interconverts ADP, ATP, and AMP to maintain this equilibrium. ADP levels in cells are typically 10% of ATP levels, and AMP levels are often less than 1% of the ATP concentration. Under such conditions, a small net change in ATP concentration due to ATP hydrolysis results in a much larger relative increase in the AMP levels because of adenylate kinase activity.

Example

Calculate the change in concentration in AMP that would occur if 8% of the ATP in an erythrocyte (red blood cell) were suddenly hydrolyzed to ADP. In erythrocytes (Table 19.2), the concentration of ATP is typically 1850 µM, the concentration of ADP is 145 µM, and the concentration of AMP is 5 µM. The total adenine nucleotide concentration is 2000 µM.

Solution

The problem can be solved using the equilibrium expression for the adenylate kinase reaction:

If 8% of the ATP is hydrolyzed to ADP, then [ATP] becomes 1850(0.92) = 1702 µM, and [AMP] + [ADP] becomes 2000 – 1702 = 298 µM, and [AMP] may be calculated from the adenylate kinase equilibrium:

Since [AMP] = 298 µM - [ADP],

[ADP] = 278 µM

[AMP] = 20 µM

Thus, an 8% decrease in [ATP] results in a 20/5 or fourfold increase in the concentration of AMP.

                Glycolysis and the citric acid cycle (to be discussed in Chapter 20) are coupled via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which “feeds” the citric acid cycle under aerobic conditions) slows down. The citric acid cycle directs electrons into the electron transport chain (for the purpose of ATP synthesis in oxidative phosphorylation ) and also provides precursor molecules for biosynthetic pathways. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated.

                Phosphofructokinase is also regulated by b-_D-fructose-2,6-bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-phosphate (Figure 19.9).

Figure 19.9 • Fructose-2,6-bisphosphate activates phosphofructokinase, increasing the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate.

Stimulation of phosphofructokinase is also achieved by decreasing the inhibitory effects of ATP (Figure 19.10). Fructose-2,6-bisphosphate increases the net flow of glucose through glycolysis by stimulating phosphofructokinase and, as we shall see in Chapter 23, by inhibiting fructose-1,6-bisphosphatase, the enzyme that catalyzes this reaction in the opposite direction.

Figure 19.10 • Fructose-2,6-bisphosphate decreases the inhibition of phosphofructo­kinase due to ATP.

Reaction 4: Cleavage of Fructose-1,6-bisP by Fructose Bisphosphate Aldolase

Fructose bisphosphate aldolase cleaves fructose-1,6-bisphosphate between the C-3 and C-4 carbons to yield two triose phosphates. The products are dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate. The reaction (Figure 19.11) has an equilibrium constant of approximately 10^-4 M, and a corresponding DG°' of +23.9 kJ/mol. These values might imply that the reaction does not proceed effectively from left to right as written. However, the reaction makes two molecules (glyceraldehyde-3-P and dihydroxyacetone-P) from one molecule (fructose-1,6-bisphosphate), and the equilibrium is thus greatly influenced by concentration. The value of DG in erythrocytes is actually -0.23 kJ/mol (see Table 19.1). At physiological concentrations, the reaction is essentially at equilibrium.

Figure 19.11 • The fructose-1,6-bisphosphate aldolase reaction.

                Two classes of aldolase enzymes are found in nature. Animal tissues produce a Class I aldolase, characterized by the formation of a covalent Schiff base intermediate between an active-site lysine and the carbonyl group of the substrate. Class I aldolases do not require a divalent metal ion (and thus are not inhibited by EDTA) but are inhibited by sodium borohydride, NaBH₄, in the presence of substrate (see A Deeper Look, page 622). Class II aldolases are produced mainly in bacteria and fungi and are not inhibited by borohydride, but do contain an active-site metal (normally zinc, Zn²⁺) and are inhibited by EDTA. Cyanobacteria and some other simple organisms possess both classes of aldolase.

                The aldolase reaction is merely the reverse of the aldol condensation well known to organic chemists. The latter reaction involves an attack by a nucleophilic enolate anion of an aldehyde or ketone on the carbonyl carbon of an aldehyde (Figure 19.12).

Figure 19.12 • An aldol condensation reaction.

The opposite reaction, aldol cleavage, begins with removal of a proton from the b-hydroxyl group, which is followed by the elimination of the enolate anion. A mechanism for the aldol cleavage reaction of fructose-1,6-bisphosphate in the Class I-type aldolases is shown in Figure 19.13a. In Class II aldolases, an active-site metal such as Zn²⁺ behaves as an electrophile, polarizing the carbonyl group of the substrate and stabilizing the enolate intermediate (Figure 19.13b).

Figure 19.13 • (a) A mechanism for the fructose-1,6-bisphosphate aldolase reaction. The Schiff base formed between the substrate carbonyl and an active-site lysine acts as an electron sink, increasing the acidity of the b -hydroxyl group and facilitating cleavage as shown. (B) In class II aldolases, an active-site Zn²⁺ stabilizes the enolate intermediate, leading to polarization of the substrate carbonyl group.

Reaction 5: Triose Phosphate Isomerase

Of the two products of the aldolase reaction, only glyceraldehyde-3-phosphate goes directly into the second phase of glycolysis. The other triose phosphate, dihydroxyacetone phosphate, must be converted to glyceraldehyde-3-phosphate by the enzyme triose phosphate isomerase (Figure 19.14).

Figure 19.14 • The triose phosphate isomerase reaction.

This reaction thus permits both products of the aldolase reaction to continue in the glycolytic pathway, and in essence makes the C-1, C-2, and C-3 carbons of the starting glucose molecule equivalent to the C-6, C-5, and C-4 carbons, respectively. The reaction mechanism involves an enediol intermediate that can donate either of its hydroxyl protons to a basic residue on the enzyme and thereby become either dihydroxyacetone phosphate or glyceraldehyde-3-phosphate (Figure 19.15).

Figure 19.15 • A reaction mechanism for triose phosphate isomerase.

Triose phosphate isomerase is one of the enzymes that have evolved to a state of “catalytic perfection,” with a turnover number near the diffusion limit (Chapter 14, Table 14.5).

                The triose phosphate isomerase reaction completes the first phase of glycolysis, each glucose that passes through being converted to two molecules of glyceraldehyde-3-phosphate. Although the last two steps of the pathway are energetically unfavorable, the overall five-step reaction sequence has a net DG°' of +2.2 kJ/mol ( K_eq » 0.43). It is the free energy of hydrolysis from the two priming molecules of ATP that brings the overall equilibrium constant close to 1 under standard-state conditions. The net DG under cellular conditions is quite negative (-53.4 kJ/mol in erythrocytes).

A Deeper Look
The Chemical Evidence for the Schiff Base Intermediate in Class I Aldolases
Fructose bisphosphate aldolase of animal muscle is a Class I aldolase, which forms a Schiff base or imine intermediate between the substrate (fructose-1,6-bisP or dihydroxyacetone-P) and a lysine amino group at the enzyme active site. The chemical evidence for this intermediate comes from studies with the aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of fructose bisphosphate aldolase with dihydroxyacetone-P and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate.         These observations are explained by the mechanism shown in the figure. NaBH4 inactivates Class I aldolases by transfer of a hydride ion (H:2) to the imine carbon atom of the enzyme-substrate adduct. The resulting secondary amine is stable to hydrolysis, and the active-site lysine is thus permanently modified and inactivated. NaBH4 inactivates Class I aldolases in the presence of either dihydroxyacetone-P or fructose-1,6-bisP, but inhibition doesn’t occur in the presence of glyceraldehyde-3-P.         Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from 14C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with 14C. Acid hydrolysis of the inactivated enzyme liberates a novel 14C-labeled amino acid, N6-dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the 14C-labeled dihydroxyacetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of 14C labeling in a case such as this facilitates the separation and identification of the telltale amino acid.

19.4  · The Second Phase of Glycolysis

The second half of the glycolytic pathway involves the reactions that convert the metabolic energy in the glucose molecule into ATP. Altogether, four new ATP molecules are produced. If two are considered to offset the two ATPs consumed in phase 1, a net yield of 2 ATPs per glucose is realized. Phase II starts with the oxidation of glyceraldehyde-3-phosphate, a reaction with a large enough energy “kick” to produce a high-energy phosphate, namely, 1,3-bisphosphoglycerate (Figure 19.16). Phosphoryl transfer from 1,3-BPG to ADP to make ATP is highly favorable. The product, 3-phosphoglycerate, is converted via several steps to phosphoenolpyruvate (PEP), another high-energy phosphate. PEP readily transfers its phosphoryl group to ADP in the pyruvate kinase reaction to make another ATP.

Figure 19.16 • The second phase of glycolysis. Carbon atoms are numbered to show their original positions in glucose.

Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase

In the first glycolytic reaction to involve oxidation-reduction, glyceraldehyde-3-phosphate is oxidized to 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase. Although the oxidation of an aldehyde to a carboxylic acid is a highly exergonic reaction, the overall reaction (Figure 19.17) involves both formation of a carboxylic-phosphoric anhydride and the reduction of NAD⁺ to NADH and is therefore slightly endergonic at standard state, with a DG°' of +6.30 kJ/mol.

Figure 19.17 • The glyceraldehyde-3-phosphate dehydrogenase reaction.

The free energy that might otherwise be released as heat in this reaction is directed into the formation of a high-energy phosphate compound, 1,3-bisphosphoglycerate, and the reduction of NAD⁺. The reaction mechanism involves nucleophilic attack by a cysteine-SH group on the carbonyl carbon of glyceraldehyde-3-phosphate to form a hemithioacetal (Figure 19.18). The hemithioacetal intermediate decomposes by hydride (H;^-) transfer to NAD⁺ to form a high-energy thioester. Nucleophilic attack by phosphate displaces the product, 1,3-bisphosphoglycerate, from the enzyme. The enzyme can be inactivated by reaction with iodoacetate, which reacts with and blocks the essential cysteine sulfhydryl.

Figure 19.18 • A mechanism for the glyceraldehyde-3-phosphate dehydrogenase reaction. Reaction of an enzyme sulfhydryl with the carbonyl carbon of glyceraldehyde-3-P forms a thiohemiacetal, which loses a hydride to NAD⁺ to become a thioester. Phosphorolysis of this thioester releases 1,3-bisphosphoglycerate.

                The glyceraldehyde-3-phosphate dehydrogenase reaction is the site of action of arsenate (AsO₄^3-), an anion analogous to phosphate. Arsenate is an effective substrate in this reaction, forming 1-arseno-3-phosphoglycerate (Figure 19.19), but acyl arsenates are quite unstable and are rapidly hydrolyzed. 1-Arseno-3-phosphoglycerate breaks down to yield 3-phosphoglycerate, the product of the seventh reaction of glycolysis. The result is that glycolysis continues in the presence of arsenate, but the molecule of ATP formed in reaction 7 ( phosphoglycerate kinase ) is not made because this step has been bypassed. The lability of 1-arseno-3-phosphoglycerate effectively uncouples the oxidation and phosphorylation events, which are normally tightly coupled in the glyceraldehyde-3-phosphate dehydrogenase reaction.

Figure 19.19 

Reaction 7: Phosphoglycerate Kinase

The glycolytic pathway breaks even in terms of ATPs consumed and produced with this reaction. The enzyme phosphoglycerate kinase transfers a phosphoryl group from 1,3-bisphosphoglycerate to ADP to form an ATP (Figure 19.20). Because each glucose molecule sends two molecules of glyceraldehyde-3-phosphate into the second phase of glycolysis and because two ATPs were consumed per glucose in the first-phase reactions, the phosphoglycerate kinase reaction “pays off” the ATP debt created by the priming reactions. As might be expected for a phosphoryl transfer enzyme, Mg²⁺ ion is required for activity, and the true nucleotide substrate for the reaction is MgADP^-. It is appropriate to view the sixth and seventh reactions of glycolysis as a coupled pair, with 1,3-bisphosphoglycerate as an intermediate. The phosphoglycerate kinase reaction is sufficiently exergonic at standard state to pull the G-3-P dehydrogenase reaction along. (In fact, the aldolase and triose phosphate isomerase are also pulled forward by phosphoglycerate kinase.) The net result of these coupled reactions is

Glyceraldehyde-3-phosphate + ADP + P_i + NAD⁺ ®
                                    3-phosphoglycerate +ATP + NADH + H⁺
                           DG°' = 212.6 kJ/mol                                                    (19.9)

Figure 19.20 • The phosphoglycerate kinase reaction.

                Another reflection of the coupling between these reactions lies in their values of DG under cellular conditions (Table 19.1). In spite of its strongly negative DG°', the phosphoglycerate kinase reaction operates at equilibrium in the erythrocyte (DG = 0.1kJ/mol). In essence, the free energy available in the phosphoglycerate kinase reaction is used to bring the three previous reactions closer to equilibrium. Viewed in this context, it is clear that ADP has been phosphorylated to form ATP at the expense of a substrate, namely, glyceraldehyde-3-phosphate. This is an example of substrate-level phosphorylation, a concept that will be encountered again. (The other kind of phosphorylation, oxidative phosphorylation, is driven energetically by the transport of electrons from appropriate coenzymes and substrates to oxygen. Oxidative phosphorylation will be covered in detail in Chapter 21). Even though the coupled reactions exhibit a very favorable DG°', there are conditions (i.e., high ATP and 3-phosphoglycerate levels) under which Equation 19.9 can be reversed, so that 3-phosphoglycerate is phosphorylated from ATP.
                An important regulatory molecule, 2,3-bisphosphoglycerate, is synthesized and metabolized by a pair of reactions that make a detour around the phosphoglycerate kinase reaction. 2,3-BPG, which stabilizes the deoxy form of hemoglobin and is primarily responsible for the cooperative nature of oxygen binding by hemoglobin (see Chapter 15), is formed from 1,3-bisphosphoglycerate by bisphosphoglycerate mutase (Figure 19.21).

Figure 19.21 • Formation and decomposition of 2,3-bisphosphoglycerate.

Interestingly, 3-phosphoglycerate is required for this reaction, which involves phosphoryl transfer from the C-1 position of 1,3-bisphosphoglycerate to the C-2 position of 3-phosphoglycerate (Figure 19.22). Hydrolysis of 2,3-BPG is carried out by 2,3-bisphosphoglycerate phosphatase . Although other cells contain only a trace of 2,3-BPG, erythrocytes typically contain 4 to 5 mM 2,3-BPG.

Figure 19.22 • The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate. The reaction is actually an intermolecular phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3-PG.

Reaction 8: Phosphoglycerate Mutase

The remaining steps in the glycolytic pathway prepare for synthesis of the second ATP equivalent. This begins with the phosphoglycerate mutase reaction (Figure 19.23), in which the phosphoryl group of 3-phosphoglycerate is moved from C-3 to C-2. (The term mutase is applied to enzymes that catalyze migration of a functional group within a substrate molecule.) The free energy change for this reaction is very small under cellular conditions ( DG = 0.83 kJ/mol in erythrocytes). Phosphoglycerate mutase enzymes isolated from different sources exhibit different reaction mechanisms.

Figure 19.23 • The phosphoglycerate mutase reaction.

As shown in Figure 19.24, the enzymes isolated from yeast and from rabbit muscle form phosphoenzyme intermediates, use 2,3-bisphosphoglycerate as a cofactor, and undergo intermolecular phosphoryl group transfers (in which the phosphate of the product 2-phosphoglycerate is not that from the 3-phosphoglycerate substrate). The prevalent form of phosphoglycerate mutase is a phosphoenzyme, with a phosphoryl group covalently bound to a histidine residue at the active site. This phosphoryl group is transferred to the C-2 position of the substrate to form a transient, enzyme-bound 2,3-bisphosphoglycerate, which then decomposes by a second phosphoryl transfer from the C-3 position of the intermediate to the histidine residue on the enzyme. About once in every 100 enzyme turnovers, the intermediate, 2,3-bisphosphoglycerate, dissociates from the active site, leaving an inactive, unphosphorylated enzyme. The unphosphorylated enzyme can be reactivated by binding 2,3-BPG. For this reason, maximal activity of phosphoglycerate mutase requires the presence of small amounts of 2,3-BPG.

Figure 19.24 • A mechanism for the phosphoglycerate mutase reaction in rabbit muscle and in yeast. Zelda Rose of the Institute for Cancer Research in Philadelphia showed that the enzyme requires a small amount of 2,3-BPG to phosphorylate the histidine residue before the mechanism can proceed. Prior to her work, the role of the phosphohistidine in this mechanism was not understood.

                A different mechanism operates in the wheat germ enzyme. 2,3-Bisphosphoglycerate is not a cofactor. Instead, the enzyme carries out intramolecular phosphoryl group transfer (Figure 19.25). The C-3 phosphate is transferred to an active-site residue and then to the C-2 position of the original substrate molecule to form the product, 2-phosphoglycerate.

Figure 19.25 • The phosphoglycerate mutase of wheat germ catalyzes an intramolecular phosphoryl transfer.

Reaction 9: Enolase

Recall that, prior to synthesizing ATP in the phosphoglycerate kinase reaction, it was necessary to first make a substrate having a high-energy phosphate. Reaction 9 of glycolysis similarly makes a high-energy phosphate in preparation for ATP synthesis. Enolase catalyzes the formation of phosphoenolpyruvate from 2-phosphoglycerate (Figure 19.26). The reaction in essence involves a dehydration—the removal of a water molecule—to form the enol structure of PEP. The DG°' for this reaction is relatively small at 1.8 kJ/mol ( K_eq = 0.5); and, under cellular conditions, DG is very close to zero. In light of this condition, it may be difficult at first to understand how the enolase reaction transforms a substrate with a relatively low free energy of hydrolysis into a product (PEP) with a very high free energy of hydrolysis. This puzzle is clarified by realizing that 2-phosphoglycerate and PEP contain about the same amount of potential metabolic energy, with respect to decomposition to P_i, CO₂, and H₂O. What the enolase reaction does is rearrange the substrate into a form from which more of this potential energy can be released upon hydrolysis. The enzyme is strongly inhibited by fluoride ion in the presence of phosphate. Inhibition arises from the formation of fluorophosphate (FPO₃^2-), which forms a complex with Mg²⁺ at the active site of the enzyme.

Figure 19.26 • The enolase reaction.

Reaction 10: Pyruvate Kinase

The second ATP-synthesizing reaction of glycolysis is catalyzed by pyruvate kinase, which brings the pathway at last to its pyruvate branch point. Pyruvate kinase mediates the transfer of a phosphoryl group from phosphoenolpyruvate to ADP to make ATP and pyruvate (Figure 19.27). The reaction requires Mg²⁺ ion and is stimulated by K⁺ and certain other monovalent cations.

Figure 19.27 • The pyruvate kinase reaction.

The corresponding K_eq at 25°C is 3.63 x 10⁵, and it is clear that the pyruvate kinase reaction equilibrium lies very far to the right. Concentration effects reduce the magnitude of the free energy change somewhat in the cellular environment, but the DG in erythrocytes is still quite favorable at -23.0 kJ/mol. The high free energy change for the conversion of PEP to pyruvate is due largely to the highly favorable and spontaneous conversion of the enol tautomer of pyruvate to the more stable keto form (Figure 19.28) following the phosphoryl group transfer step.

Figure 19.28 • The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer followed by an enol-keto tautomerization. The tautomerization is spontaneous ( DG°' » -35-40 kJ/mol) and accounts for much of the free energy change for PEP hydrolysis.

                The large negative DG of this reaction makes pyruvate kinase a suitable target site for regulation of glycolysis. For each glucose molecule in the glycolysis pathway, two ATPs are made at the pyruvate kinase stage (because two triose molecules were produced per glucose in the aldolase reaction). Because the pathway broke even in terms of ATP at the phosphoglycerate kinase reaction (two ATPs consumed and two ATPs produced), the two ATPs produced by pyruvate kinase represent the “payoff” of glycolysis —a net yield of two ATP molecules.
                Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Hormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phosphorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher K_m for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the gluconeogenesis pathway (to be described in Chapter 23), instead of going on through glycolysis and the citric acid cycle (or fermentation routes). A suggested active-site geometry for pyruvate kinase, based on NMR and EPR studies by Albert Mildvan and colleagues, is presented in Figure 19.29. The carbonyl oxygen of pyruvate and the g -phosphorus of ATP lie within 0.3 nm of each other at the active site, consistent with direct transfer of the phosphoryl group without formation of a phosphoenzyme intermediate.

Figure 19.29 • A mechanism for the pyruvate kinase reaction, based on NMR and EPR studies by Albert Mildvan and colleagues. Phosphoryl transfer from phosphoenolpyruvate (PEP) to ADP occurs in four steps: (a) a water on the Mg²⁺ ion coordinated to ADP is replaced by the phosphoryl group of PEP; (b) Mg²⁺ dissociates from the a-P of ADP; (c) the phosphoryl group is transferred; and (d) the enolate of pyruvate is protonated. (Adapted from Mildvan, A., 1979. Advances in Enzymology 49:103-126.)

19.5  · The Metabolic Fates of NADH and Pyruvate —The Products of Glycolysis

In addition to ATP, the products of glycolysis are NADH and pyruvate. Their processing depends upon other cellular pathways. NADH must be recycled to NAD⁺, lest NAD⁺ become limiting in glycolysis. NADH can be recycled by both aerobic and anaerobic paths, either of which results in further metabolism of pyruvate. What a given cell does with the pyruvate produced in glycolysis depends in part on the availability of oxygen. Under aerobic conditions, pyruvate can be sent into the citric acid cycle (also known as the tricarboxylic acid cycle; see Chapter 20), where it is oxidized to CO₂ with the production of additional NADH (and FADH₂). Under aerobic conditions, the NADH produced in glycolysis and the citric acid cycle is reoxidized to NAD⁺ in the mitochondrial electron transport chain (Chapter 21).

19.6  · Anaerobic Pathways for Pyruvate

Under anaerobic conditions, the pyruvate produced in glycolysis is processed differently. In yeast, it is reduced to ethanol; in other microorganisms and in animals, it is reduced to lactate. These processes are examples of fermentation—the production of ATP energy by reaction pathways in which organic molecules function as donors and acceptors of electrons. In either case, reduction of pyruvate provides a means of reoxidizing the NADH produced in the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis (Figure 19.30). In yeast, alcoholic fermentation is a two-step process. Pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase in an essentially irreversible reaction. Thiamine pyrophosphate is a required cofactor for this enzyme. The second step, the reduction of acetaldehyde to ethanol by NADH, is catalyzed by alcohol dehydrogenase (Figure 19.30). At pH 7, the reaction equilibrium strongly favors ethanol. The end products of alcoholic fermentation are thus ethanol and carbon dioxide. Alcoholic fermentations are the basis for the brewing of beers and the fermentation of grape sugar in wine making. Lactate produced by anaerobic microorganisms during lactic acid fermentation is responsible for the taste of sour milk and for the characteristic taste and fragrance of sauerkraut, which in reality is fermented cabbage.

Figure 19.30 • (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD⁺ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (b) In oxygen-depleted muscle, NAD⁺ is regenerated in the lactate dehydrogenase reaction.

Lactate Accumulates Under Anaerobic Conditions in Animal Tissues

In animal tissues experiencing anaerobic conditions, pyruvate is reduced to lactate. Pyruvate reduction occurs in tissues that normally experience minimal access to blood flow (e.g., the cornea of the eye) and also in rapidly contracting skeletal muscle. When skeletal muscles are exercised strenuously, the available tissue oxygen is consumed, and the pyruvate generated by glycolysis can no longer be oxidized in the TCA cycle. Instead, excess pyruvate is reduced to lactate by lactate dehydrogenase (Figure 19.30). In anaerobic muscle tissue, lactate represents the end of glycolysis. Anyone who exercises to the point of consuming all available muscle oxygen stores knows the cramps and muscle fatigue associated with the buildup of lactic acid in the muscle. Most of this lactate must be carried out of the muscle by the blood and transported to the liver, where it can be resynthesized into glucose in gluconeogenesis. Moreover, because glycolysis generates only a fraction of the total energy available from the breakdown of glucose (the rest is generated by the TCA cycle and oxidative phosphorylation ), the onset of anaerobic conditions in skeletal muscle also means a reduction in the energy available from the breakdown of glucose.

19.7  · The Energetic Elegance of Glycolysis

The elegance of nature’s design for the glycolytic pathway may be appreciated through an examination of Figure 19.31. The standard-state free energy changes for the 10 reactions of glycolysis and the lactate dehydrogenase reaction (Figure 19.31a) are variously positive and negative and, taken together, offer little insight into the coupling that occurs in the cellular milieu. On the other hand, the values of DG under cellular conditions (Figure 19.31b) fall into two distinct classes. For reactions 2 and 4 through 9, DG is very close to zero, so that these reactions operate essentially at equilibrium. Small changes in the concentrations of reactants and products could “push” any of these reactions either forward or backward. By contrast, the hexokinase, phosphofructokinase, and pyruvate kinase reactions all exhibit large negative DG values under cellular conditions. These reactions are thus the sites of glycolytic regulation. When these three enzymes are active, glycolysis proceeds and glucose is readily metabolized to pyruvate or lactate. Inhibition of the three key enzymes by allosteric effectors brings glycolysis to a halt. When we consider gluconeogenesis—the biosynthesis of glucose—in Chapter 23, we will see that different enzymes are used to carry out reactions 1, 3, and 10 in reverse, effecting the net synthesis of glucose. The maintenance of reactions 2 and 4 through 9 at or near equilibrium permits these reactions (and their respective enzymes!) to operate effectively in either the forward or reverse direction.

Figure 19.31 • A comparison of free energy changes for the reactions of glycolysis (step 1 = hexokinase) under (a) standard-state conditions and (b) actual intracellular conditions in erythrocytes. The values of DG°' provide little insight into the actual free energy changes that occur in glycolysis. On the other hand, under intracellular conditions, seven of the glycolytic reactions operate near equilibrium (with DG near zero). The driving force for glycolysis lies in the hexokinase (1), phosphofructokinase (3), and pyruvate kinase (10) reactions. The lactate dehydrogenase (step 11) reaction also exhibits a large negative DG under cellular conditions.

19.8  · Utilization of Other Substrates in Glycolysis

Figure 19.32 • Mannose, galactose, fructose, and other simple metabolites can enter the glycolytic pathway.

The glycolytic pathway described in this chapter begins with the breakdown of glucose, but other sugars, both simple and complex, can enter the cycle if they can be converted by appropriate enzymes to one of the intermediates of glycolysis. Figure 19.32 shows the mechanisms by which several simple metabolites can enter the glycolytic pathway. Fructose, for example, which is produced by breakdown of sucrose, may participate in glycolysis by at least two different routes. In the liver, fructose is phosphorylated at C-1 by the enzyme fructokinase :

D-Fructose + ATP^4- ® D-fructose-1-phosphate^2- + ADP^3- + H⁺           (19.10)

Subsequent action by fructose-1-phosphate aldolase cleaves fructose-1-P in a manner like the fructose bisphosphate aldolase reaction to produce dihydroxyacetone phosphate and D-glyceraldehyde:

D-Fructose-1-P^2- ® D-glyceraldehyde + dihydroxyacetone phosphate^2-      (19.11)

Dihydroxyacetone phosphate is of course an intermediate in glycolysis. D-Glyceraldehyde can be phosphorylated by triose kinase in the presence of ATP to form D-glyceraldehyde-3-phosphate, another glycolytic intermediate.

                In the kidney and in muscle tissues, fructose is readily phosphorylated by hexokinase, which, as pointed out above, can utilize several different hexose substrates. The free energy of hydrolysis of ATP drives the reaction forward:

D-Fructose + ATP^4- ® D-gructose-6-phosphate^2- + ADP^3- H⁺       (19.12)

Fructose-6-phosphate generated in this way enters the glycolytic pathway directly in step 3, the second priming reaction. This is the principal means for channeling fructose into glycolysis in adipose tissue, which contains high levels of fructose.

The Entry of Mannose into Glycolysis

Another simple sugar that enters glycolysis at the same point as fructose is mannose, which occurs in many glycoproteins, glycolipids, and polysaccharides (Chapter 7). Mannose is also phosphorylated from ATP by hexokinase, and the mannose-6-phosphate thus produced is converted to fructose-6-phosphate by phosphomannoisomerase .

D-Mannose + ATP^4- ® D-mannose-6-phosphate^2- + ADP^3- H⁺                 (19.13)

D-Mannose-6-phosphate^2- + ATP^4- ® D-fructose-6-phosphate^2-                 (19.14)

The Special Case of Galactose

A somewhat more complicated route into glycolysis is followed by galactose, another simple hexose sugar. The process, called the Leloir pathway after Luis Leloir, its discoverer, begins with phosphorylation from ATP at the C-1 position by galactokinase:

 D-Galactose + ATP^4- ® D-galactose-1-phosphate^2- +ADP^3- + H⁺               (19.15)

Galactose-1-phosphate is then converted into UDP-galactose (a sugar nucleotide) by galactose-1-phosphate uridylyltransferase (Figure 19.33), with concurrent production of glucose-1-phosphate and consumption of a molecule of UDP-glucose.

Figure 19.33 • Galactose metabolism via the Leloir pathway.

The uridylyltransferase reaction proceeds via a “ping-pong” mechanism (Figure 19.34) with a covalent enzyme-UMP intermediate. The glucose-1-phosphate produced by the transferase reaction is a substrate for the phosphoglucomutase reaction (Figure 19.33), which produces glucose-6-phosphate, a glycolytic substrate. The other transferase product, UDP-galactose, is converted to UDP-glucose by UDP-glucose-4-epimerase. The combined action of the uridylyltransferase and epimerase thus produces glucose-1-P from galactose-1-P, with regeneration of UDP-glucose.

Figure 19.34 • The galactose-1-phosphate uridylyltransferase reaction involves a “ping-pong” kinetic mechanism.

                A rare hereditary condition known as galactosemia involves defects in galactose-1-P uridylyltransferase that render the enzyme inactive. Toxic levels of galactose accumulate in afflicted individuals, causing cataracts and permanent neurological disorders. These problems can be prevented by removing galactose and lactose from the diet. In adults, the toxicity of galactose appears to be less severe, due in part to the metabolism of galactose-1-P by UDP-glucose pyrophosphorylase, which apparently can accept galactose-1-P in place of glucose-1-P (Figure 19.35). The levels of this enzyme may increase in galactosemic individuals, in order to accommodate the metabolism of galactose.

Figure 19.35 • The UDP-glucose pyrophosphorylase reaction.

Lactose Intolerance

A much more common metabolic disorder, lactose intolerance, occurs commonly in most parts of the world (notable exceptions being some parts of Africa and northern Europe ). Lactose intolerance is an inability to digest lactose because of the absence of the enzyme lactase in the intestines of adults. The symptoms of this disorder, which include diarrhea and general discomfort, can be relieved by eliminating milk from the diet.

Glycerol Can Also Enter Glycolysis

Glycerol is the last important simple substance whose ability to enter the glycolytic pathway must be considered. This metabolite, which is produced in substantial amounts by the decomposition of triacylglycerols (see Chapter 24) can be converted to glycerol-3-phosphate by the action of glycerol kinase and then oxidized to dihydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase, with NAD⁺ as the required coenzyme (Figure 19.36). The dihydroxyacetone phosphate thereby produced enters the glycolytic pathway as a substrate for triose phosphate isomerase.

Figure 19.36