· web viewunder standard conditions, aldol condensation is favored since the standard free energy...
TRANSCRIPT
Glycolysis
What is glycolysis?
Glycolysis comes from the Greek glykys meaning “sweet” and lysis meaning splitting.
Glycolysis is the sequence of reaction that converts glucose into pyruvate with the concomitant
production of a relatively small amount of adenosine triphosphate (ATP). It is the initial process
of most carbohydrate catabolism.
Glucose is a key metabolite in human metabolism, and we will spend a good bit of time on the
various pathways that are concerned with the utilization, storage, and regeneration of glucose.
The first step in the degradation of glucose is glycolysis, which breaks down glucose to pyruvate.
The main purpose of glycolysis is the generation of energy (ATP). A modest amount of ATP is
produced in glycolysis directly, but much more ATP is formed downstream of glycolysis through
the complete oxidation of pyruvate. An alternative pathway for complete glucose breakdown is
the hexose monophosphate shunt, which produces NADPH rather than ATP. Both ATP and
NADPH are needed in every cell, and accordingly both glycolysis and the hexose
monophosphate shunt are ubiquitous.
Glycogen is a polymeric storage form of glucose, not unlike starch, which is found in plants.
Glycogen is most abundant in the liver and in striated muscle, although some is found in other
tissues also. Glycogen is synthesized when glucose supply is high, and its degradation helps to
maintain the blood glucose level when we are fasting. When glycogen is depleted, more glucose
is synthesized from scratch in gluconeogenesis. This pathway’s most important substrates are
amino acids, which are obtained either from a protein-rich diet—for example, when we feast on
meat exclusively—or, during starvation, from breakdown of cellular protein, mainly in skeletal
muscle. Gluconeogenesis occurs in the liver and in the kidneys.
1
HISTORY OF GLYCOLYSIS
In years 1854 to 1864, Louis Pasteur established that fermentation is caused by microorganisms.
• 1897 – Eduard Buchner demonstrated that cell-free yeast extracts can carry out this process.
• In years 1905 to 1910, Arthur Harden and William Young discovered:
– Inorganic phosphate is required for fermentation and is incorporated into fructose-1-6-
bisphosphate
– A cell-free yeast extract has a nondialyzable heat-labile fraction (zymase) and a dialyzable
heat-stable fraction (cozymase).
During the 1930s, the efforts of several German biochemists, including Gustav Embden, Otto
Meyerhof, and Jacob Parnas, determined that glycolysis involves 10 steps, each one catalyzed
by a different enzyme.
OVER VIEW OF GLYCOLYSIS
● Two phases of glycolysis. 1. Preparatory phases 2. Pay off phases
● There are ten steps, 7 reversible; 3 irreversible (1, 3&10)
● In the first phase, 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 glyceraldehydes
3-phosphate into two molecules of pyruvate.
● Phase 1 consumes two ATP.
●T he later stages of glycolysis result in the production of 4 ATP and 2 NADH.
● The net is 4 – 2 = 2 ATP produced per molecule of glucose.
● All glycolysis reactions occur in the cytosol.
● Two triose compounds are isomerized and oxidized to retrieve ATP & NADH via
glyceraldehydes 3-phosphate dehydrogenase.
● The pathway concludes with a strong, irreversible, ATP-producing step to make pyruvate.
● Glucose is stored in big polymers like starch or glycogen
● When energy demand increases, it can be released quickly and used to produce ATP either
aerobically (using oxygen) or anaerobically (fermentation)
2
● Glucose is also a versatile molecule for building the basic carbon skeleton of other molecules
like fatty acids, amino acids etc.
GLYCOLYTIC PATHWAY
3
REACTION OF GLYCOSIS
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.
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 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.
Reaction 2: Isomerization of glucose-6-P to fructose-6-P by phosphoglucose isomerase
4
G6P is then rearranged into fructose 6-phosphate (F6P) by glucose phosphate isomerase.
Fructose can also enter the glycolytic pathway by phosphorylation at this point.
The change in structure is an isomerization, in which the G6P has been converted to F6P. The
reaction requires an enzyme, phosphohexose isomerase, to proceed. This reaction is freely
reversible under normal cell conditions. However, it is often driven forward because of a low
concentration of F6P, which is constantly consumed during the next step of glycolysis. Under
conditions of high F6P concentration, this reaction readily runs in reverse. This phenomenon can
be explained through Le Chatelier's Principle. Isomerization to a keto sugar is necessary for
carbanion stabilization in the fourth reaction step (below).
α-D-Glucose 6-phosphate (G6P) Phosphoglucose isomerasean isomerase β-D-Fructose 6-phosphate (F6P)
Reaction 3: Phosphorylation of fructose- 6-P to fructose-1,6-BP byphosphofructokinase-1
The energy expenditure of another ATP in this step is justified in 2 ways: The glycolytic process
(up to this step) is now irreversible, and the energy supplied destabilizes the molecule. Because
the reaction catalyzed by Phosphofructokinase 1 (PFK-1) is coupled to the hydrolysis of ATP, an
energetically favorable step, it is, in essence, irreversible, and a different pathway must be used
to do the reverse conversion during gluconeogenesis. This makes the reaction a key regulatory
point (see below). This is also the rate-limiting step.
5
Furthermore, the second phosphorylation event is necessary to allow the formation of two
charged groups (rather than only one) in the subsequent step of glycolysis, ensuring the
prevention of free diffusion of substrates out of the cell.
The same reaction can also be catalyzed by pyrophosphate-dependent phosphofructokinase (PFP
or PPi-PFK), which is found in most plants, some bacteria, archea, and protists, but not in
animals. This enzyme uses pyrophosphate (PPi) as a phosphate donor instead of ATP. It is a
reversible reaction, increasing the flexibility of glycolytic metabolism.[8] A rarer ADP-dependent
PFK enzyme variant has been identified in archaean species.
Reaction 4: Cleavage of fructose-1,6-BP into glyceraldehyde-3-P and dihydroxyacetone-P
The splitting of fructose-1,6-BP into the triose phosphates glyceraldehyde-3-P (GAP) and
dihydroxyacetone-P (DHAP) is the reaction that puts the lysis in glycolysis (lysis means
splitting) as shown in figure 13. The enzyme responsible for this cleavage reaction 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).
6
Reaction 5: Isomerization of dihydroxyacetone-P to glyceraldehyde-3-P bytriose 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.
7
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. 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.
Reaction 7: Substrate level phosphorylation to generate ATP in the conversion of1,3-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. 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.
8
Reaction 8: Phosphoryl shift in 3-phosphyglycerate to form 2-phosphoglycerate byphosphoglycerate 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 20.
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 22), 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.
9
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.
Reaction 10: Substrate level phosphorylation to generate ATP in the conversion ofphosphoenolpyruvate 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 (figure 23). 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 describedlater.
What are the two substrate level phosphorylation reactions 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.
10
ENERGETIC COUNT
Step
Reaction Phosphorylated /reduced coenzyme
ATP count
1 Glucose + ATP giucose- 6- phosphate + ADP 1 ATP used -1 ATP3 Fructose- 6- phosphate + ATP fructose- 1,6- bisphosphate 1 ATP used -1 ATP6 2 × glyceraldehyde-3- phosphate + 2×NAD+ + 2 × Pi
2× 1,3- bisphosphoglycerate + 2 ×NADH + H+ 2 ×NADH + H+ + 6 ATP
7 2× 1,3- bisphosphoglycerate + 2 ADP 2× 3- phosphoglucerate + 2 ATP
2 ATP produced + 2 ATP
10 2 phosphoenolpyruvate 2 × pyruvate + 2 ATP 2 ATP produced + 2 ATPTotal energy gain + 8 ATP
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.
First, 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).
Second, 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.
11
12
Entrance of glycogen, starch, disaccharides and hexoses
13
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.
Regulation of glycolysis occurs at three points of the pathway. These correspond to the steps
with the largest negative free energy changes (i.e. most exergonic - negative ΔG). The magnitude
of the ΔG for these steps makes them essentially irreversible. The most important point of
control is at the reaction catalyzed by hexokinase, phosphofructokinase and pyruvate kinase
reactions.
Hexokinase and glucokinase
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 by 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 rates of glycolytic flux in all cells by limiting substrate availability.
14
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 2. Since
blood glucose levels are maintained around 5mM, significant levels of glucose phosphorylation
by glucokinase only occur 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. 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.
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
15
potent allosteric regulator of PFK-1 activity (discussed in lecture 35). The PFK-1 reaction in
glycolysis is irreversible and functions as one of three metabolic “valves” that controls flux
through the pathway (the other two are the hexokinase and pyruvate kinase reactions). Figure 4
illustrates that AMP
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 5 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).
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 7, 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.
(Allosteric Enzymes: Allosteric means “other site”. These enzymes have two receptor sites. One
site fits the substrate like other enzymes. The other site fits an inhibitor or activator molecule.
Molecules that bind to allosteric sites are called effectors or modulators. Binding of a substance
to one binding site increases or decrease the another site. Effectors may be positive or negative.)
16
An enzyme that can be in two states, active and inactive, in the first instance, the process is relatively
normal, where the enzyme binds to its binding Site and carries out its reaction. In the second instance, the
enzyme is unable to attach itself to the binding Site. This is due to a modulator molecule which alters the
shape of the binding Site, making it inactive.allosteric enzyme
Pyruvate kinase
Pyruvate kinase is the third regulated enzyme of glycolysis. Like PFK, pyruvate kinase is
regulated both by allosteric effectors and by covalent modification (phosphorylation). Pyruvate
kinase is activated by F-1,6-BP in the liver, a second example of feedforward stimulation. ATP
and alanine (a biosynthetic product of pyruvate) act as allosteric inhibitors of pyruvate kinase.
17
Summary: Regulation of the glycolytic pathway
18
The flux through the glycolytic pathway must be adjusted in response to conditions both inside and
outside the cell. The rate of conversion of glucose into pyruvate is regulated to meet two major cellular
needs: (1) the production of ATP, generated by the degradation of glucose, and (2) the provision of
building blocks for synthetic reactions, such as the formation of fatty acids. In metabolic pathways,
enzymes catalyzing essentially irreversible reactions are potential sites of control. In glycolysis, the
reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are virtually irreversible;
hence, these enzymes would be expected to have regulatory as well as catalytic roles. In fact, each of
them serves as a control site. Their activities are regulated by the reversible binding of allosteric effectors
or by covalent modification. In addition, the amounts of these important enzymes are varied by the
regulation of transcription to meet changing metabolic needs. The time required for reversible allosteric
control, regulation by phosphorylation, and transcriptional control is typically in milliseconds, seconds,
and hours, respectively.
Phosphofructokinase is the most prominent regulatory enzyme in glycolysis, but it is not the only one.
Hexokinase, the enzyme catalyzing the first step of glycolysis, is inhibited by its product, glucose 6-
phosphate. High concentrations of this molecule signal that the cell no longer requires glucose for energy,
for storage in the form of glycogen, or as a source of biosynthetic precursors, and the glucose will be left
in the blood. For example, when phosphofructokinase is inactive, the concentration of fructose 6-
phosphate rises. In turn, the level of glucose 6-phosphate rises because it is in equilibrium with fructose 6-
phosphate. Hence, the inhibition of phosphofructokinase leads to the inhibition of hexokinase. However,
the liver, in keeping with its role as monitor of blood-glucose levels, possesses a specialized isozyme of
hexokinase called glucokinase that is not inhibited by glucose 6-phosphate. Glucokinase phosphorylates
glucose only when it is abundant because it has about a 50-fold affinity for glucose than does hexokinase.
The role of glucokinase is to provide glucose 6-phosphate for the synthesis of glycogen, a storage form of
glucose (Section 21.4), and for the formation of fatty acids (Section 22.1). The low glucose affinity of
glucokinase in the liver gives the brain and muscles first call on glucose when its supply is limited,
whereas it ensures that glucose will not be wasted when it is abundan
Pyruvate kinase, the enzyme catalyzing the third irreversible step in glycolysis, controls the
outflow from this pathway. This final step yields ATP and pyruvate, a central metabolic
intermediate that can be oxidized further or used as a building block. Several isozymic forms of
19
pyruvate kinase (a tetramer of 57-kd subunits) encoded by different genes are present in
mammals: the L type predominates in liver, and the M type in muscle and brain. The L and M
forms of pyruvate kinase have many properties in common. Both bind phosphoenolpyruvate
cooperatively. Fructose 1,6-bisphosphate, the product of the preceding irreversible step in
glycolysis, activates both isozymes to enable them to keep pace with the oncoming high flux of
intermediates. ATP allosterically inhibits both the L and the M forms of pyruvate kinase to slow
glycolysis when the energy charge is high. Finally, alanine (synthesized in one step from
pyruvate, Section 24.2.2) also allosterically inhibits the pyruvate kinases—in this case, to signal
that building blocks are abundant.
The isozymic forms differ in their susceptibility to covalent modification. The catalytic
properties of the L form—but not of the M form—are also controlled by reversible
phosphorylation (Figure 16.21). When the blood-glucose level is low, the glucagon-triggered
cyclic AMP cascade (Section 15.1.5) leads to the phosphorylation of pyruvate kinase, which
diminishes its activity. These hormone-triggered phosphorylations, like that of the bifunctional
enzyme controlling the levels of fructose 2,6-bisphosphate, prevent the liver from consuming
glucose when it is more urgently needed by brain and muscle (Section 30.3). We see here a
clear-cut example of how isoenzymes contribute to the metabolic diversity of different organs.
We will return to the control of glycolysis after considering gluconeogenesis.
20