Bioc 460 - Dr.

Miesfeld 3/24/04

Glycolysis 1 & 2 (complete text with figures)

Lectures 24, 25

Key Concepts
- Overview of the Glycolytic Pathway
• Glycolysis generates a small amount of ATP
• Preview of the ten enzyme-catalyzed reactions of glycolysis
- Stage 1: ATP Investment
• Reaction 1: Hexokinase
• Reaction 2: Phosphoglucose isomerase
• Reaction 3: Phosphofructokinase
• Reaction 4: Aldolase
• Reaction 5: Triose phosphate isomerase
- Stage 2: ATP Earnings
• Reaction 6: Glyceraldehyde-3-P dehydrogenase
• Reaction 7: Phosphoglycerate kinase (substrate level phosphorylation)
• Reaction 8: Phosphoglycerate mutase
• Reaction 9: Enolase
• Reaction 10: Pyruvate kinase (substrate level phosphorylation)
- Regulation of the Glycolytic Pathway
• Glucokinase is a molecular sensor of high glucose levels
• Allosteric control of phosphofructokinase activity
• Supply and demand of glycolytic intermediates
- Metabolic Fate of Pyruvate

KEY CONCEPT QUESTIONS IN GLYCOLYSIS:

What are the two substrate level phosphorylation reactions in glycolysis?
How do substrate availability and enzyme activity levels control glycolytic flux?
Why is muscle lactate dehydrogenase activity required for short bursts of intense exercise?

Biochemical Applications of Glycolysis:

Lactose intolerance is caused by reduced

levels of the digestive enzyme lactase which is
required to hydrolyze the disaccharide sugar
lactose to form glucose and galactose. Purified
lactase enzyme is commercially available and
can be taken as a pill at meal time to reduce
symptoms that occur when bacteria of the
Lactobacillus family convert lactose to methane
and hydrogen gases in the small intestine.

OVERVIEW OF THE GLYCOLYTIC PATHWAY

Glycolysis is considered one of the core metabolic pathways in nature for three primary reasons, 1)
glycolytic enzymes are highly conserved amongst all living organisms, suggesting it is an ancient
pathway, 2) glycolysis is the primary pathway for ATP generation under anaerobic conditions and
in cells lacking mitochondria such as erythrocytes, and 3) metabolites of glycolysis are precursors
for a large number of interdependent pathways, including mitochondrial ATP synthesis. The word
glycolysis is derived from the Greek glykys, meaning sweet, and lysis which means to split or
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break, referring to the splitting of one molecule of glucose into two molecules of pyruvate. We will
focus on two aspects of glycolysis, 1) the enzymatic reactions that convert glucose to pyruvate,
and 2) the role of glycolysis in providing precursors, especially pyruvate, for other metabolic
pathways in humans.

Let’s begin by answering the four questions in our guidebook that pertain to glycolysis:

1. What does glycolysis accomplish for the cell?

• Generates a small amount of ATP which is critical under anaerobic conditions.
• Generates pyruvate, a precursor to acetyl CoA, lactate, and ethanol (in yeast).

2. What is the overall net reaction of glycolysis?

Glucose + 2NAD+ + 2ADP + 2 Pi  2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O
ΔGº’ = -35.8 kJ/mol
3. What are the key regulated enzymes in glycolysis?
Hexokinase – commitment step in glycolysis, inhibited by glucose-6-P (product)
Phosphofructokinase – activated by AMP (low energy charge) and F-2,6-BP, inhibited by ATP
(high energy charge) and citrate (citrate cycle intermediate)
Pyruvate kinase - activated by AMP and fructose-1,6-bisphosphate (feed forward), inhibited by
ATP and acetyl CoA (excess energy source)

4. What are examples of glycolysis in real life?

A deficiency in the hexokinase-related enzyme glucokinase, leads to a rare form of diabetes which
is caused by the inability of liver and pancreatic cells to phosphorylate glucose inside cells when
blood glucose levels are elevated.

We first need to see where glycolysis fits into our metabolic map. As shown in figure 1, glycolysis
sits at the top of a set of four interconnected pathways that together are responsible for the
complete oxidation of glucose to CO2 and H2O by the following reaction:

Glucose (C6H12O6) + 6O2  6CO2 + 6H2O ΔGº’ = -2,840 kJ/mol

ΔG = -2,937 kJ/mol

Glycolysis generates a small amount of ATP Figure 1.

As shown in figure 2, glycolysis takes place entirely in the
cytosol, whereas, pyruvate oxidation to CO2 and H2O occurs in
the mitochondrial matrix and requires the citrate cycle, electron
transport chain and oxidative phosphorylation. Acetyl CoA is
the primary metabolite in energy conversion pathways inside
the mitochondrial matrix and is also the major breakdown
product of fatty acid oxidation (lecture 22). Glycolysis does not
require oxygen and therefore, can function under anaerobic
conditions, however, complete oxidation of glucose to CO2 and
H2O requires oxygen.

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Figure 2.

The combined reactions of glycolysis, the citrate cycle, electron transport chain and oxidative
phosphorylation, yield 32 ATP from glucose oxidation. Glycolysis alone yields only 2 ATP out of
the total 32 (6%). Therefore, it is the oxidation of pyruvate (and acetyl CoA) in the mitochondria
that generates the majority of ATP (94%) for most cells in an organism.

Preview of the ten enzyme-catalyzed reactions of glycolysis

Figure 3 shows the molecular structures of glucose and pyruvate to illustrate that the six carbons
and six oxygens present in glucose are stoichiometrically conserved by glycolysis in the two
molecules of pyruvate that are produced.

Figure 3.

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The ten enzymatic reactions of glycolysis are summarized in figure 4 and involve primarily bond
rearrangements brought about by enzymes that catalyze phosphoryl transfer reactions,
isomerizations, an aldol cleavage, an oxidation, and a dehydration. There is no net loss of carbon
or oxygen atoms. Because of the requirement for ATP hydrolysis in the initial reactions, followed
by ATP synthesis in the later reactions, glycolysis is divided into two stages, Stage 1: ATP
investment (reactions 1-5) and Stage 2: ATP earnings (reactions 6-10).

Figure 4.

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Figure 5 summarizes the glycolytic pathway showing that for every mole of glucose entering
glycolysis, two moles of glyceraldehyde-3-P are metabolized to pyruvate, and in the process,
generating a net 2 ATP and 2 NADH. The "nodes" in this wiring diagram represent metabolites in
the glycolytic pathway.Key reaction steps are highlighted along with the corresponding enzymes.
Figure 5.

One way to understand how a pathway is regulated is to examine free energy changes (ΔGº’ and
ΔG) that take place in each reaction to identify steps that are critical in driving the overall pathway
towards product formation. The free energy changes for the ten glycolytic reactions are listed in
Figure 6 including both the ΔGº’ of each reaction which is measured in the laboratory under
standard conditions, and the calculated ΔG value using ΔG = ΔGº’ +RT•ln(mass action ratio)
based on measured metabolite concentrations in erythrocytes under steady-state conditions.
Figure 6.

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The reactions catalyzed by the enzymes hexokinase, phosphofructokinase and pyruvate kinase
have large negative ΔG values and are therefore considered irreversible reactions under
physiological conditions. Although several of the glycolytic reactions are shown to have positive
ΔG values, it is likely that the mass action ratios for these reactions do not represent actual
metabolite concentrations under conditions of high metabolic flux. A good example of this is
reaction 5 catalyzed by the enzyme triose phosphate isomerase which has an estimated ΔG of
+2.5 kJ/mol, but is nevertheless pulled to the right due to the rapid depletion of glyceraldehyde-3-P
by the highly favorable reactions in stage 2 of glycolysis. Indeed, the net ΔG value for glycolysis in
erythrocytes under steady-state conditions is overwhelmingly negative (-76.5 kJ/mol), confirming
that the glycolytic pathway is highly favorable.

STAGE 1: ATP INVESTMENT

Stage 1 of glycolysis includes five enzymatic reactions that accomplish two tasks. First, using ATP
as the phosphate donor, they create phosphorylated compounds that are negatively charged
and cannot diffuse out of the cell. These phosphorylated metabolites are highly specific substrates
for glycolytic enzymes and are the precursors to the high energy compounds 1,3-bisphoglycerate
and phosphoenolpyruvate in stage 2 that are used to generate ATP by substrate level
phosphorylation. Second, the aldolase reaction in step 4 splits the six carbon fructose-1,6-BP
compound into two halves creating glyceraldehyde-3-P and dihydroxyacetone-P, the latter of
which is quickly isomerized to form a second molecule of glyceraldehyde-3-P.

• Reaction 1: Phosphorylation of glucose by hexokinase or glucokinase

The first reaction in glycolysis serves to activate glucose for catabolism by attaching a phosphate
group to the C6 position to generate glucose-6-P as illustrated in figure 7. This is the first of two
ATP investment steps in stage 1 of glycolysis and uses the free energy released from ATP
hydrolysis to drive the phosphoryl transfer reaction. Figure 8 shows the pyranose ring structure of
glucose and glucose-6-P.
Figure 7.

Figure 8.

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Two enzymes catalyze this phosphorylation reaction, hexokinase which is found in all cells, and
glucokinase which is present primarily in liver and pancreatic cells. Hexokinase has a broad range
of substrate specificities and also phosphorylates mannose and fructose, whereas, glucokinase is
highly specific for glucose. Hexokinase activity is inhibited by the product of the reaction, glucose-
6-P, which accumulates in cells when flux through the glycolytic pathway is restricted. As
described later, glucokinase has a much lower affinity for glucose and is not feedback inhibited by
glucose-6-P. These enzymatic properties, along with its selective expression in specific cell types,
facilitate the function of glucokinase as a metabolic “sensor” of high blood glucose levels.

Hexokinase binds glucose through an induced fit mechanism that excludes H2O from the enzyme
active site and brings the phosphoryl group of ATP into close proximity with the C6 carbon of
glucose. The molecular structure of yeast hexokinase in the presence and absence of glucose
suggests that two domains of the enzyme are like jaws that clamp down on the substrate through a
large conformational change (see figure 9). Figure 10 illustrates the induced fit mechanism of
hexokinase and also shows that glucose-6-P inhibition of hexokinase activity is mediated by
binding of glucose-6-P to an effector site in the N-terminal domain of the protein.
Figure 9.

Figure 10.

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• Reaction 2: Isomerization of glucose-6-P to fructose-6-P by phosphoglucose isomerase

As shown in figure 11, phosphoglucose isomerase (phosphohexose isomerase) interconverts an
aldose (glucose-6-P) and a ketose (fructose-6-P) through a multi-step pathway that involves
opening and closing of the ring structure. The reaction is readily reversible (ΔGº’ = 1.7kJ/mol) and
controlled by the level of glucose-6-P and fructose-6-P in the cell.

Figure 11.

• Reaction 3: Phosphorylation of fructose-6-P to fructose-1,6-BP by phosphofructokinase

Reaction 3 is the second ATP investment reaction in glycolysis and involves the coupling of ATP
hydrolysis to a phosphoryl transfer reaction that is catalyzed by the enzyme phosphofructokinase.
The reaction is essentially irreversible with a large decrease in standard free energy
(ΔGº’ = -14.2kJ/mol) and serves as major regulatory site in the pathway through allosteric control
of phosphofructokinase activity in response to the energy charge of the cell. As shown in figure 12,
phosphorylation of fructose-6-P generates fructose-1,6-bisphosphate (fructose-1,6-BP) which will
form two different triose phosphates when cleaved in reaction 4. Note that a bisphosphate
compound contains two phosphates on different atoms (C1 and C6), whereas, a diphosphate
compound such as ADP, contains two phosphates covalently linked to each other.

Figure 12.

• Reaction 4: Cleavage of fructose-1,6-BP into glyceraldehyde-3-P and dihydroxyacetone-P by

aldolase
The splitting of fructose-1,6-BP into the triose phosphates glyceraldehyde-3-P and
dihydroxyacetone-P is the reaction that puts the lysis in glycolysis (lysis means splitting) as shown
in figure 13. The enzyme responsible for this cleavage reaction between the C3 and C4 carbons in
fructose-1,6-BP is aldolase (fructose bisphosphate aldolase), which performs the reverse of an
aldol condensation in the context of the glycolytic pathway. The aldolase reaction illustrates the
important difference between ΔGº’ and ΔG values. Under standard conditions, aldol condensation
is favored since the standard free energy for the cleavage reaction is highly positive (ΔGº’ =
+23.9kJ/mol).

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Figure 13.

• Reaction 5: Isomerization of dihydroxyacetone-P to glyceraldehyde-3-P by triose phosphate

isomerase
The production of dihydroxyacetone-P by aldol cleavage of fructose-1,6-BP in reaction 4 above
creates a slight problem because glyceraldehyde-3-P, not dihydroxyacetone-P, is the substrate for
reaction 6 in the glycolytic pathway. This dilemma is solved by the enzyme triose phosphate
isomerase which converts the ketose dihydroxyacetone-P to the aldose glyceraldehyde-3-P in an
isomerization reaction that is similar to reaction 2 (conversion of glucose-6-P to fructose-6-P),
albeit in reverse (this time we need an aldose formed from a ketose). Figure 14 shows the reaction
catalyzed by triose phosphate isomerase which completes stage 1 of glycolysis, and at the
expense of 2ATPs, produces two moles of glyceraldehyde-3-P for every mole of glucose that is
phosphorylated in reaction 1.
Figure 14.

Figure15.
The enzyme triose phosphate isomerase was introduced earlier in
the course as an example of an α/β protein structure called a TIM
barrel which was first identified in the molecular structure of triose
phosphate isomerase, thus the name TIM. As shown in figure 15,
the triose substrate sits in the enzyme active site which is formed
by the β strands and is located in the center of the protein.

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STAGE 2: ATP EARNINGS

Stage 2 of glycolysis is where the real action takes place in glycolysis, and because two moles of
glyceraldehyde-3-P enter stage 2 for every mole of glucose metabolized, stage 2 is twice as
exciting as stage 1. Three key features of stage 2 reactions need to be pointed out before we
begin. First, two substrate level phosphorylation reactions catalyzed by the enzymes
phosphoglycerate kinase and pyruvate kinase generate a total of 4ATPs (net yield of 2ATP) in
stage 2 of glycolysis. Second, an oxidation reaction catalyzed by glyceraldehyde-3-P
dehydrogenase generates 2 NADH molecules that can be shuttled into the mitochondria to
produce more ATP by oxidative phosphorylation. Third, reaction 10 is an irreversible reaction
that must be bypassed in gluconeogenesis by two separate enzymatic reactions catalyzed by
pyruvate carboxylase and phosphoenolpyruvate carboxykinase.

• Reaction 6: Oxidation and phosphorylation of glyceraldehyde-3-P to form

1,3-bisphosphoglycerate by glyceraldehyde-3-P dehydrogenase
The glyceraldehyde-3-P dehydrogenase reaction is a critical step in glycolysis because it uses the
energy released from oxidation of glyceradehyde-3-P to drive a phosphoryl group transfer reaction
using inorganic phosphate (Pi) to produce 1,3-bisphosphoglycerate (figure 16). This coupled
reaction requires the coenzyme NAD+ and includes the formation of an acyl thioester intermediate
within the enzyme active site to conserve the oxidation energy. Since NAD+ is required for the
oxidation step in this reaction, NAD+ must be continually replenished within the cytosol to maintain
flux through glycolysis. This is accomplished aerobically in the mitochondrial matrix by the electron
transport chain, or anaerobically in the cytosol by the enzyme lactate dehydrogenase which
converts pyruvate to lactate. Anaerobic fermentation in yeast regenerates NAD+ using the enzyme
alcohol dehydrogenase which converts pyruvate to CO2 and ethanol.

Figure 16.

The enzyme mechanism for glyceraldehyde-3-P dehydrogenase has been worked out and is
illustrated in figure 17. Glyceraldehyde-3-P binds to the enzyme active site which contains an
associated NAD+ coenzyme molecule which will function as an electron acceptor and then reacts
with the sulfhydryl group of a cysteine residue in the enzyme active site to form a hemithioacetal
intermediate. The aldehyde group is then dehydrogenated by an oxidation reaction in which a
hydride ion (:H-) is transferred from the enzyme bound glyceraldehyde-3-P to the nicotinamide ring
of NAD+ to produce NADH, a second hydrogen is removed from the substrate and released into
solution (H+). This oxidation reaction results in the formation of a high energy enzyme-substrate
complex called a thioester intermediate which is then subject to nucleophilic attack by an incoming
phosphate ion leading to product release (1,3-bisphosphoglycerate) and regeneration of the active
enzyme.

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Figure 17.

Importantly, formation of 1,3-bisphosphoglycerate by this coupled oxidation-phosphorylation

reaction generates a metabolite with a standard free energy of hydrolysis that is higher than ATP
hydrolysis. This difference in free energies is harnessed by the enzyme phosphoglycerate kinase
in reaction 7 to drive the synthesis of ATP by a mechanism called substrate level phosphorylation.
Figure 18 shows that another glycolytic intermediate, phosphoenolpyruvate, has a standard free
energy of phosphate hydrolysis that exceeds both 1,3-bisphosphoglycerate and ATP. This may
explain why the acronym for phosphoenolypyruvate, “PEP”, is so easy to remember (a “peppy”
personality is full of life and vigor). PEP provides the energy in reaction 10 to generate another
molecule of ATP during the formation of pyruvate. Note that glucose-6-P has a standard free
energy of phosphate hydrolysis that is lower than that of ATP, which explains the required ATP
investment reaction in step 1 of glycolysis.

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Table 18.

• Reaction 7: Substrate level phosphorylation to generate ATP in the conversion of

1,3-bisphosphoglycerate to 3-phosphoglycerate by phosphoglycerate kinase
Phosphoglycerate kinase catalyzes the payback reaction in glycolysis because it replaces the 2
ATP that were used in stage 1 to prime the glycolytic pathway. As shown in figure 19, the high
phosphoryl transfer energy present in the substrate is used to phosphorylate ADP to form ATP by
the mechanism of substrate level phosphorylation, leading to the conversion of 1,3-
bisphosphoglycerate to 3-phosphoglycerate. Remember that two moles of 1,3-bisphophoglycerate
are formed from every mole of glucose, therefore this reaction occurs twice and generates
2ATP/glucose.

Figure 19.

The molecular structure of phosphoglycerate kinase is similar to hexokinase in that it has two lobes
(jaws) that each bind one of the substrates (ADP-Mg2+ or 1,3-bisphosphoglycerate) leading to a
large conformational change in the enzyme that brings the substrates close together and excludes
H2O from the active site. The structures of phosphoglycerate kinase in the open and closed
conformations are shown in figure 20.

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Figure 20.

The bioenergetics of reaction 7 emphasize two important concepts we have presented in the
chapter. First, reaction 6 (glyceraldehyde-3-P dehydrogenase) and reaction 7 (phosphoglycerate
kinase) are coupled reactions in that the large change in standard free energy of reaction 7 (ΔGº’
= -18.9 kJ/mol) pulls the less favorable reaction 6 (ΔGº’ = +6.3 kJ/mol) to the right through the
shared intermediate 1,3-bisphosphoglcerate as shown below:

(Rxn 6) Glyceraldehyde-3-P + Pi + NAD+  1,3-bisphosphoglycerate + NADH + H+

ΔGº’ = +6.3 kJ/mol ΔG = -1.3 kJ/mol

(Rxn 7) 1,3-bisphosphoglycerate + ADP  3-phosphoglycerate + ATP

ΔGº’ = -18.9 kJ/mol ΔG = +0.1 kJ/mol
(Coupled) Glyceraldehyde-3-P + Pi + ADP + NAD  3-phosphoglycerate + ATP + NADH + H+
+

ΔGº’ = -12.6 kJ/mol ΔG = -1.2 kJ/mol

Second, the actual change in free energy for each of these two reactions is very close to zero
(ΔG = -1.3 kJ/mol, ΔG = +0.1 kJ/mol), and therefore both reactions are in fact reversible inside
the cell. Again, this difference in ΔGº’ and ΔG is due to the mass action ratio which takes into
account the actual concentrations of substrates and products that exist in the cell. Why is the
reversibility of these two reactions important? The answer is that when flux through
gluconeogenesis is high (lecture 23), these two glycolytic reactions can be reversed and thus
quickly respond to changing conditions in the cell.

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• Reaction 8: Phosphoryl shift in 3-phosphyglycerate to form 2-phosphoglycerte by

phosphoglycerate mutase
The purpose of reaction 8 is to generate a compound, 2-phosphoglycerate, that can be converted
to phosphoenolpyruvate in the next reaction, in preparation for a second substrate level
phosphorylation that generates ATP earnings in step 10. The phosphoglycerate mutase reaction
is shown in figure 21.
Figure 21.

The mechanism of this highly reversible reaction is illustrated in figure 22 where it can be seen to
require a phosphoryl transfer from a phosphorylated histidine residue (His-P) located in the
enzyme active site. In step 1, the substrate 3-phosphoglycerate binds to the enzyme active site
and is phosphorylated in the C2 position by a transfer reaction involving the His-P group. This type
of substrate interaction with the enzyme is non-covalent and referred to as a substrate•enzyme
complex. Phosphoryl transfer from the histidine residue to the C2 atom of the 3-phosphoglycerate
creates the short-lived intermediate 2,3-bisphosphoglycerate (BPG). In the second step of the
reaction, the C3 phosphate is transferred back to the histidine residue of the enzyme to regenerate
His-P, leading to the release of 2-phosphoglycerate and binding of a new molecule of 3-
phosphoglycerate in the third step. Note that the BPG formed in step 1 can diffuse out of the
active site resulting in dephosphorylated enzyme, and you may remember that in red blood cells
BPG has an important role in regulating oxygen binding to hemoglobin in red blood cells. When
BPG leaves the active site without re-phosphorylating the His group, the enzyme can only be
activated when trace amounts of BPG diffuse back into the active site.
Figure 22.

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• Reaction 9: Dehydration of 2-phosphoglycerate to form phosphoenolpyruvate by enolase

In this penultimate step in glycolysis, a dehydration reaction catalyzed by the enzyme enolase
converts 2-phosphoglycerate, a molecule with only moderate phosphoryl transfer potential, to
phosphoenolpyruvate (figure 23), which we have already seen has extremely high phosphoryl
transfer potential. It is this high phosphoryl transfer potential in phosphoenolpyruvate that is
harnessed in the last reaction in glycolysis to form ATP.
Figure 23.

It is interesting that the change in standard free energy for this reaction is relatively small (ΔGº’ =
+1.7) kJ/mol), meaning that the overall metabolic energy available from 2-phosphoglycerate and
phosphoenolpyruvate is similar. However, when enolase converts 2-phosphoglycerate to
phosphoenolpyruvate, it traps the phosphate group in an unstable enol form, resulting in a
dramatic increase in the phosphoryl transfer potential of the triose sugar. The standard free
energy change for phosphate hydrolysis in 2-phosphoglycerate is ΔGº’ = -16 kJ/mol, whereas for
phosphoenolpyruvate it is an incredible ΔGº’ = -62 kJ/mol.

• Reaction 10: Substrate level phosphorylation to generate ATP in the conversion of

phosphoenolpyruvate to pyruvate by pyruvate kinase
In this reaction, the high phosphoryl transfer potential of phosphoenolpyruvate is used by the
enzyme pyruvate kinase to generate pyruvate, the end product of glycolysis, and 2 ATP are
formed for every glucose molecule entering the pathway. This is the second of two substrate level
phosphorylation reactions in glycolysis that couples energy released from phosphate hydrolysis
(ΔGº’ = -62 kJ/mol) to that of ATP synthesis (ΔGº’ = +30.5 kJ/mol) as shown in figure 24. Unlike
phosphoenolpyruvate, pyruvate is a stable compound in cells that is utilized by many other
metabolic pathways as will be described later.

Figure 24.

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REGULATION OF THE GLYCOLYTIC PATHWAY

Irreversible reactions in metabolic pathways are called rate-limiting steps because the level of
enzyme activity can be low even when substrate levels are high. Rate-limiting enzymes in
metabolic pathways serve as regulated “valves” that are opened or closed in response to cellular
conditions. As illustrated in figure 25, reversible steps in glycolysis and gluconeogenesis operate
in both pathways, whereas, irreversible steps have actual changes in free energies (ΔG) that are
highly negative and require pathway-specific enzymes.

Figure 25.

Glucokinase is a molecular sensor of high glucose levels

Four hexokinase genes have been identified in humans (hexokinase I-IV), all of which are capable
of converting glucose to glucose-6-P at the expense of ATP hydrolysis (step 1 of glycolysis). We
have already described one of these, hexokinase I (reaction 1), which has a high affinity for
substrate (Km for glucose is ~0.1mM), is expressed in all tissues, phosphorylates a variety of
hexose sugars, and is inhibited by the product of the reaction, glucose-6-P. In contrast, hexokinase
IV, also known as glucokinase, has a low affinity for substrate (Km for glucose is ~10mM), is highly
specific for glucose, is expressed primarily in liver and pancreatic cells and is not inhibited to
glucose-6-P. This difference in tissue expression and glucose affinity between hexokinase and
glucokinase plays an important role in controlling blood glucose levels, which ultimately controls
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rates of glycolytic flux in all cells by limiting substrate availability. As suggested by the different Km
values of hexokinase and glucokinase for glucose, substrate saturation curves for these two
enzymes look markedly different as shown in figure 26. Since blood glucose levels are maintained
around 5mM, significant levels of glucose phosphorylation by glucokinase only occurs under
conditions of high glucose, such as after consuming a carbohydrate-rich meal. Moreover, since
glucokinase is not inhibited by glucose-6-P, it is able to continue functioning even if flux through
glycolysis cannot keep up with product formation.

Figure 26.

The role of glucokinase in liver cells is to trap the extra glucose that is available from the diet so
that it can be stored as glycogen for an energy source later. By being active in liver cells only when
glucose concentrations exceed normal limits (>5mM), glucokinase ensures that the liver is the
major sink for dietary glucose, and at the same time, is able to efficiently remove glucose from the
blood to help restore normal blood glucose concentrations.

Another important function of glucokinase is to act as a glucose sensor in pancreatic β cells

where glucokinase enzyme levels are activated by increased glucose import mediated by glucose
transporter proteins (GLUT proteins). As shown in figure 27, when the concentration of glucose in
the blood is elevated, glucose import into pancreatic β cells leads to higher glucokinase enzyme
levels resulting in increased flux through glycolysis and net ATP synthesis. This increase in ATP
levels causes inhibition of ATP-sensitive K+ channels, membrane depolarization, and activation of
voltage-gated Ca2+ channels. Elevated levels of intracellular Ca2+ triggers fusion of insulin-
containing vesicles with the plasma membrane and subsequent release of insulin into the blood.
This intracellular signaling pathway links glucose uptake in pancreatic cells with insulin release.

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Figure 27.

The importance of pancreatic glucokinase in insulin secretion was confirmed using transgenic mice
in which the glucokinase gene was specifically deleted in pancreatic β cells. These glucokinase-
deficient mice were defected in glucose-stimulated insulin secretion and became hyperglycemic,
eventually developing diabetic symptoms due to chronic elevated blood glucose. Well over 100
mutations in the human glucokinase gene have been identified and it is now known that a form of
type II diabetes in humans, called maturity-onset diabetes of the young (MODY2), is caused by
defects in pancreatic glucokinase activity.

Allosteric control of phosphofructokinase activity

Of all the enzymes in glycolysis, phosphofructokinase is the best characterized because of its vital
role in controlling flux through the pathway. There are actually two phosphofructokinase isozymes
(distinct genes that encode proteins with similar functions), phosophofructokinase-1 (PFK-1) which
catalyzes reaction 3 in glycolysis, and phosphofructokinase-2 (PFK-2) a bifunctional enzyme that
catalyzes the synthesis of fructose-2,6-bisophosphate (F-2,6-BP), a potent allosteric regulator of
PFK-1 activity. The PFK-1 reaction in glycolysis is essentially irreversible and functions as one of
three metabolic “valves” (the other two being reactions catalyzed by hexokinase and pyruvate
kinase) that controls flux through the pathway. Figure 28 illustrates that AMP, ADP and F-2,6-BP
are activators of PFK-1 activity, and ATP and citrate function as inhibitors.
Figure 28.

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PFK-1 is an allosteric enzyme that exists as a tetramer (a dimer of dimers) in either of two
conformations, the inactive T state or active R state, analogous to the hemoglobin tetramer. The
equilibrium between T and R states in a cell is controlled by allosteric effector molecules which
bind to a regulatory site outside of the substrate binding pocket. ATP and citrate are negative
effector molecules of PFK-1 which stabilize the T state, whereas, AMP, ADP and F-2,6-BP are
positive effector molecules that stabilize the R state. Figure 29 shows how the allosteric regulators
ATP, AMP and F-2,6-BP alter the PFK-1 reaction rate as a function of substrate concentration
(fructose-6-P).
Figure 29.

Figure 30 shows the molecular structure of PFK-1 as a monomer with reaction products fructose-
1,6-BP and ADP in the active site, and as a dimer in the active R conformation with ADP bound to
allosteric effector site. Note that the allosteric effector site is far removed from the catalytic site
and in fact maps to the interface between two PFK-1 subunits.

Figure 30.

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Studies have shown that ATP binds with equal affinity to the catalytic site regardless of the T or R
state conformation of PFK-1. However, ATP binding to the allosteric effector site is highest when
the protein is in the T state which functions to decrease fructose-6-P binding to the catalytic site.
As illustrated in figure 31, AMP binding to the allosteric effector site serves to stabilize the R state,
and thereby stimulates the production of fructose-1,6-BP by ATP-mediated phosphoryl transfer.
This equilibrium shift between the T or R states is modulated by the energy charge of the cell such
that high ATP concentrations (high energy charge) increase the pool of PFK-1 molecules in the T
state, whereas, high AMP concentrations shifts the pool to more PFK-1 molecules in the direction
of the R state.

Figure 31.

Supply and demand of glycolytic intermediates

We have so far described the starting point for glycolysis as glucose import into the cell from the
circulatory system via GLUT transport proteins, followed by hexokinase or glucokinase catalyzed
phosphorylation to produce glucose-6-P. Glucose can also be derived from glycogen which we
mentioned in lecture 21 is the storage form of glucose polymers in liver and muscle cells. When
glucose is needed as energy source for muscle cells undergoing contraction, glycogen is degraded
by the enzyme glycogen phosphorylase to produce glucose-1-P (lecture 33).

Plants can’t make glycogen, but they do store glucose as large polymeric molecules in starch
granules. Animals that eat plants such as ourselves, break down dietary starch into the
disaccharide maltose by an enzyme in saliva called α-amylase. As shown in figure 32, maltose is
then cleaved by the enzyme maltase in the intestine to produce two molecules of glucose. Other
dietary sources of carbohydrates are the disaccharides sucrose and lactose which are cleaved by
the hydrolytic enzymes sucrase and lactase, respectively, into the monosaccharides glucose,
fructose and galactose (figure 32). Sucrose is common table sugar, and fructose is present at high
levels in many types of fruits and vegetables. Glycerol is a three carbon metabolite that is a
component of triglycerides which contain three covalently linked fatty acid molecules. A class of
hydrolytic enzymes known as lipases, cleave off the fatty acids from the triglyceride molecule to
release glycerol which can enter the glycolytic pathway. The fatty acid components of triglycerides
are metabolized in mitochondria by the fatty acid oxidation pathway to produce acetyl CoA.
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Figure 32.

The conversion of lactose, sometimes called milk sugar, to galactose and glucose by the enzyme
lactase may be familiar to you if you are lactose-intolerant (lactose-sensitive). Individuals with
this condition experience considerable discomfort from associated intestinal symptoms (excessive
flatulence and diarrhea) when lactose-containing foods such as dairy products are eaten. The
human gene for lactase is expressed at high levels in infants to digest lactose in breast milk,
however, lactase expression normally declines in adults with the notable exception of individuals of
Scandinavian descent. The intestinal symptoms of lactose intolerance are caused by the activity of
naturally occurring anaerobic bacteria in the human intestine from the genus Lactobacillus
which ferment the undigested lactose to lactate, producing hydrogen (H2) and methane (CH4)
gases as side products. Diarrhea becomes a problem if the amount of unhydrolyzed lactose is so
high that it osmotically increases water flow into the intestine. The simplest way to prevent these
symptoms is to not eat food products containing lactose, for example, by eating soy milk products

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rather than dairy products. Fortunately, biotechnology has provided a way to have your ice cream
and eat it too through the industrial production of purified lactase enzyme.
Fructose, galactose and glycerol enter the glycolytic pathway through a variety of routes,
many of which require additional enzymatic reactions as shown in figure 33. Glycerol for example
enters glycolysis through a two step reaction requiring the enzymes glycerol kinase and glycerol
phosphate dehydrogenase to form the glycolytic intermediate dihydroxyacetone-P. Some
metabolites enter glycolysis through a single phosphorylation reaction such as fructose in muscle
cells which is converted to fructose-6-P by the enzyme hexokinase. Fructose metabolism in liver
cells, however, is more complicated in that dietary fructose is first converted to fructose-1-P by the
enzyme fructokinase. Fructose-1-P is cleaved by fructose-1-P aldolase to generate
dihydroxyacetone-P and glyceraldehyde. Dihydroxyacetone-P is isomerized to glyceraldehyde-3-P
by the glycolytic enzyme trios phosphate isomerase, and glyceraldehyde is phosphorylated by
triose kinase to produce glyceraldehyde-3-P.
Figure 33.

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Fructose metabolism uses the same number of invested ATP molecules as does glucose in both
muscle (1 ATP) and liver cells (2 ATP) and is a good source of dietary carbohydrate. In fact, high
fructose corn syrup is the most common added sweetener to processed foods. However, for
individuals with a genetic disease called fructose intolerance, fructose in the diet can be
extremely toxic. Fructose intolerance is due to deficiencies in the enzyme fructose-1-P
aldolase, also called aldolase B. People with fructose intolerance cannot eat foods containing
fructose because it leads to the build-up of fructose-1-P which has no alternate metabolic fates in
the absence of aldolase B. Since Pi turnover in cells is required to continually synthesize large
amounts of ATP by oxidative phosphorylation, the accumulation of fructose-1-P acts as a Pi “sink”
by tying up available phosphate in the liver that would normally be recycled by ATP hydrolysis.
Under these conditions, liver cells are quickly depleted of ATP causing ATP-dependent cation
membrane pumps to shut down, resulting in cell lysis and liver damage.
Glycolytic intermediates also serve important roles in anabolic pathways by providing
carbon skeletons for biosynthesis. Besides playing a central role in gluconeogenesis, numerous
glycolytic intermediates are used in a variety of pathways including amino acid biosynthesis, and in
the formation glycolipids and glycoproteins as illustrated in figure 34. This figure also shows that
glycolytic intermediates generate two important regulatory molecules, 2,3-bisphosphoglycerate
(BPG) and fructose-2,6-bisphosphate (F-2,6-BP), through one-step phosphorylation reactions.
Figure 34.

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METABOLIC FATE OF PYRUVATE

The end product of glycolysis, pyruvate, is metabolized in one of three ways depending on the
organism and the availability of oxygen as illustrated in figure 35. Under aerobic conditions, the
majority of pyruvate is metabolized in the mitochondria to acetyl CoA, and ultimately to CO2 and
H2O which are the products of the citrate cycle and electron transport chain. Aerobic metabolism
in the mitochondrial matrix is responsible for the majority of ATP synthesis in cells that depend
on the oxidation of metabolic fuel for energy conversion (plants obtain energy for ATP synthesis
from sunlight). However, under anaerobic conditions, such as occurs in muscle cells during
strenuous exercise, or in erythrocytes which lack mitochondria, pyruvate is converted to lactate
(the ionized form of lactic acid) by the enzyme lactate dehydrogenase. A variety of
microorganisms also convert pyruvate to lactate under anaerobic conditions, for example,
Lactobacillus bulgaricus, a strain of bacteria used in the dairy industry to produce foods such
as yogurt and cheese. The third fate of pyruvate occurs in microorganisms such as yeast which
utilize alcoholic fermentation to convert pyruvate to CO2 and ethanol using the enzymes pyruvate
decarboxylase and alcohol dehydrogenase, respectively.
Figure 35.

These three pyruvate pathways are responsible for regenerating the coenzyme NAD+ required to
maintain flux through the glyceraldehyde-3-P dehydrogenase reaction in glycolysis. This metabolic
requirement for NAD+ in glycolysis can be seen in individuals who have defects in the enzyme
lactate dehydrogenase, a genetic disease called lactate dehydrogenase deficiency (LDHA).
These patients cannot maintain moderate levels of exercise due to an inability to utilize glycolysis
to produce ATP needed for muscle contraction under anaerobic conditions. The lactate
dehydrogenase reaction is shown in figure 36 where it can be seen that pyruvate reduction to
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lactate is coupled to the oxidation of NADH to produce NAD+. When lactate dehydrogenase is not
fully functional, NADH oxidation does not occur at a high enough rate to sustain glycolysis. This
causes the muscle cells to quickly run out of ATP leading to fatigue and even muscle damage if
anaerobic conditions persist. We will discuss how lactate production in athletes is normally
controlled by the liver when we look at metabolic integration in lectures 40 and 41.
Figure 36.

ANSWERS TO KEY CONCEPT QUESTIONS IN GLYCOLYSIS:

Substrate level phosphorylation reactions generate ATP by capturing the high phosphate transfer
potential of the substrate in a coupled reaction with ADP. The enzymes phosphoglycerate kinase
and pyruvate kinase catalyze the two substrate level phosphorylation reactions in stage 2 of
glycolysis. Since two molecules of glyceraldehyde-3-P are generated for every molecule of
glucose metabolized, each of these substrate level phosphorylation reactions yield 2ATP/glucose.
Phosphoglycerate kinase (reaction 7) provides the payback of 2ATP invested in stage 1 of
glycolysis to generate fructose-1,6-BP, whereas, reaction 10 catalyzed by pyruvate kinase
represents the ATP earnings step in the pathway by generating 2 net ATP.

Substrate availability and enzyme activity levels control glycolytic flux by regulating reaction rates.
Three of the ten glycolytic reactions are essentially irreversible under cellular conditions (ΔG<<0)
and are catalyzed by enzymes that are subject to regulation, whereas, the other seven reactions
are reversible (ΔG~0) and respond to substrate availability. Phosphofructokinase activity is
controlled by the negative allosteric effector molecules ATP and citrate which signal high energy
charge in the cell, and by the positive allosteric effector molecules ADP and F-2,6-BP which signal
low energy charge. Since phosphofructokinase is a key rate-limiting enzyme in the glycolytic
pathway, the activity level of phosphofructokinase has a major affect on glycolytic flux.
Glucokinase is subject to both types of control in that it has a low affinity for glucose and is only
active when glucose levels are high, moreover, glucokinase enzyme levels are stimulated by
glucose signaling in pancreatic b cells.

Cytosolic NAD+ is required to maintain glycolytic flux through the glyceraldehyde-3-P

dehydrogenase reaction. Glycolysis is the primary source of ATP when oxygen is limiting, such as
during intense strenuous exercise, and also in erythrocytes which lack mitochondria. The cytosolic
enzyme lactate dehydrogenase regenerates NAD+ by oxidizing NADH and is highly active under
anaerobic conditions due to the build-up of pyruvate which cannot be metabolized to acetyl CoA.
Individuals that have defects in muscle lactate dehydrogenase cannot sustain strenuous exercise
because NAD+ is not regenerated quickly enough to maintain glycolytic flux and supply the
necessary ATP for muscle contraction. Yeast alcohol dehydrogenase performs the same function
of regenerating NAD+ to maintain glycolytic flux during the process of alcoholic fermentation.
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