glycolysis
TRANSCRIPT
GLYCOLYSIS CONCERNS
Although glycolysis is a nearly universal process, the fate of its
end product, pyruvate, may vary in different organisms or even
in different tissues.
In the presence of oxygen, the most common situation in
multicellular organisms and many unicellular ones, pyruvate is
metabolized to carbon dioxide and water through the citric acid
cycle and the electron transport chain.
In the absence of oxygen, fermentation generates a lesser
amount of energy; pyruvate is converted, or fermented, into
lactic acid in lactic acid fermentation or into ethanol in alcoholic
fermentation.
Lactic acid production takes place in skeletal muscle when
energy needs outpace the ability to transport oxygen.
Glycolysis is a catabolic pathway in the cytoplasm that is found
in almost all organisms—irrespective of whether they live
aerobically or anaerobically.
In eukaryotic cells, glycolysis takes place in the cytosol. This
pathway can be thought of as comprising three stages
Stage 1, which is the conversion of glucose into fructose 1, 6-
bisphosphate, consists of three steps: a phosphorylation, an
isomerization, and a second phosphorylation reaction.
The strategy of these initial steps in glycolysis is to trap the
glucose in the cell and form a compound that can be readily
cleaved into phosphorylated three-carbon units.
Stage 2 is the cleavage of the fructose 1,6-bisphosphate into
two three-carbon fragments. These resulting three-carbon units
are readily interconvertible.
In stage 3, ATP is harvested when the three carbon fragments
are oxidized to pyruvate.
Glycolysis involves ten individual steps, including three
isomerizations and four phosphate transfers. The only redox
reaction takes place in step [6].
[1] Glucose, which is taken up by animal cells from the blood
and other sources, is first phosphorylated to glucose 6-
phosphate, with ATP being consumed. The glucose 6-phosphate
is not capable of leaving the cell.
[2] In the next step, glucose 6-phosphate is isomerized into
fructose 6-phosphate.
[3] Using ATP again, another phosphorylation takes place, giving
rise to fructose 1,6-bisphosphate. Phosphofructokinase is the
most important key enzyme in glycolysis.
[4] Fructose 1,6-bisphosphate is broken down by aldolase into
the C3 compounds glyceraldehyde3-phosphate (also known as
glyceral3-phosphate) and glycerone3-phosphate
(dihydroxyacetone 3-phosphate).
[5] The latter two products are placed in fast equilibrium by
triosephosphate isomerase.
[6] Glyceraldehyde 3-phosphate is now oxidized by
glyceraldehyde-3-phosphate dehydrogenase, with NADH + H+
being formed. In this reaction, inorganic phosphate is taken up
into the molecule (substrate-level phosphorylation; and 1,3-
bisphosphoglycerate is produced. This intermediate contains a
mixed acid–anhydride bond, the phosphate part of which is at a
high chemical potential.
[7] Catalyzed by phosphoglycerate kinase, this phosphate
residue is transferred to ADP, producing 3-phosphoglycerate and
ATP. The ATP balance is thus once again in equilibrium.
[8] As a result of shifting of the remaining phosphate residue
within the molecule, the isomer 2-phosphoglycerate is formed.
[10] In the last step, pyruvate kinase transfers this residue to
ADP. The remaining enol pyruvate is immediately rearranged
into pyruvate, which is much more stable.
SOME HIGHLIGHTS
*1*Glucose is phosphorylated by ATP to form glucose 6-
phosphate. This step is notable for two reasons: (1) glucose 6-
phosphate cannot diffuse through the membrane, because of its
negative charges, and (2) the addition of the phosphoryl group
begins to destabilize glucose, thus facilitating its further
metabolism.
The glucose-induced structural changes are significant in two
respects. First, the environment around the glucose becomes
much more nonpolar, which favors the donation of the terminal
phosphoryl group of ATP. Second, the substrate-induced
conformational changes within the kinase enable it to
discriminate against H2O as a substrate. That means it blocks
the access of water (from the solvent), which might otherwise
enter the active site and attack (hydrolyze) the
phosphoanhydride bonds (esp., the γ phosphoryl group) of ATP
forming ADP and Pi. In other words, a rigid kinase would
necessarily also be an ATPase.
Like the other nine enzymes of glycolysis, hexokinase is a
soluble, cytosolic protein.
Hexokinase, like adenylate kinase and all other kinases, requires
Mg 2 + (or another divalent metal ion such as Mn2+) for activity.
The divalent metal ion forms a complex with ATP.
Kinetic studies of NMP kinases, as well as many other enzymes
having ATP or other nucleoside triphosphates as a substrate,
reveal that these enzymes are essentially inactive in the absence
of divalent metal ions such as magnesium (Mg2+) or manganese
(Mn2+), but acquire activity on the addition of these ions.
The metal is not a component of the active site. Rather,
nucleotides such as ATP bind these ions, and it is the metal ion-
nucleotide complex that is the true substrate for the enzymes.
Essentially all nucleoside triphosphates are present as NTP-
Mg2+ complexes.
(1) The magnesium ion neutralizes some of the negative charges
present on the polyphosphate chain, reducing nonspecific ionic
interactions between the enzyme and the polyphosphate group
of the nucleotide.
(2) The interactions between the magnesium ion and the oxygen
atoms in the phosphoryl group hold the nucleotide in well-
defined conformations that can be specifically bound by the
enzyme.
(3) The magnesium ion provides additional points of interaction
between the ATP-Mg2+ complex and the enzyme, thus
increasing the binding energy.
**In some cases, such as the DNA polymerases, side chains
(often aspartate and glutamate residues) of the enzyme can
bind directly to the magnesium ion. In other cases, the enzyme
interacts indirectly with the magnesium ion through hydrogen
bonds to the coordinated water molecules (Figure 9.50). Such
interactions have been observed in adenylate kinases bound to
ATP analogs.
Hexokinase is present in all cells of all organisms. Hepatocytes
also contain a form of hexokinase called hexokinase IV or
glucokinase, which differs from other forms of hexokinase in
kinetic and regulatory properties.
Two enzymes that catalyze the same reaction but are encoded
in different genes are called isozymes.
*2* The enzyme phosphohexose isomerase (phosphoglucose
isomerase) catalyzes the reversible isomerization of glucose 6-
phosphate, an aldose, to fructose 6-phosphate, a ketose.
The enzyme must first open the six-membered ring of glucose 6-
phosphate, catalyze the isomerization, and then promote the
formation of the five-membered ring of fructose 6-phosphate.
*3* Phosphorylation of Fructose 6-Phosphate to Fructose 1,6-
Bisphosphate, phosphofructokinase-1 (PFK-1) catalyzes the
transfer of a phosphoryl group from ATP to fructose 6-
phosphate to yield fructose 1,6-bisphosphate.
This enzyme is called PFK-1 to distinguish it from a second
enzyme (PFK-2) that catalyzes the formation of fructose 2,6-
bisphosphate from fructose 6-phosphate in a separate pathway.
The PFK-1 reaction is essentially irreversible under cellular
conditions.
Phosphofructokinase-1 is a regulatory enzyme, one of the most
complex known.
PFK1 is the major point of regulation in glycolysis. The activity of
PFK-1 is increased whenever the cell’s ATP supply is depleted or
when the ATP breakdown products, ADP and AMP (particularly
the latter), are in excess. The enzyme is inhibited whenever the
cell has ample ATP and is well supplied by other fuels such as
fatty acids. In some organisms, fructose 2,6-bisphosphate is a
potent allosteric activator of PFK-1.
The enzyme fructose 1,6-bisphosphate aldolase, often called
simply aldolase, catalyzes a reversible aldol condensation.
*4* Fructose 1,6-bisphosphate is cleaved to yield two different
triose phosphates, glyceraldehyde 3-phosphate (GAP) , an
aldose, and dihydroxyacetone phosphate (DHAP) , a ketose.
Although the aldolase reaction has a strongly positive standard
free-energy change in the direction of fructose 1,6-bisphosphate
cleavage, at the lower concentrations of reactants present in
cells, the actual free-energy change is small and the aldolase
reaction is readily reversible.
Only one of the two triose phosphates formed by aldolase,
glyceraldehyde 3-phosphate, can be directly degraded in the
subsequent steps of glycolysis.
*5* The other product, dihydroxyacetone phosphate, is rapidly
and reversibly converted to glyceraldehyde 3-phosphate by the
fifth enzyme of the sequence, triose phosphate isomerase.
This reaction is rapid and reversible. At equilibrium, 96% of the
triose phosphate is dihydroxyacetone phosphate.
*5* TIM catalyzes the transfer of a hydrogen atom from carbon
1 to carbon 2 in converting dihydroxyacetone phosphate into
glyceraldehyde 3-phosphate, an intramolecular oxidation-
reduction.
Two features of this enzyme are noteworthy. First, TIM displays
great catalytic prowess. Indeed, the k cat/K M ratio for
isomerization of glyceraldehyde 3-phosphate is 2 × 108 M-1 s-1,
which is close to the diffusion-controlled limit. In other words,
the rate-limiting step in catalysis is the diffusion-controlled
encounter of substrate and enzyme. TIM is an example of a
kinetically perfect enzyme. Second, TIM suppresses an
undesired side reaction, the decomposition of the enediol
intermediate into methyl glyoxal and inorganic phosphate.
The payoff phase of glycolysis includes the energy-conserving
phosphorylation steps in which some of the free energy of the
glucose molecule is conserved in the form of ATP.
One molecule of glucose yields two molecules of glyceraldehyde
3-phosphate; both halves of the glucose molecule follow the
same pathway in the payoff phase of glycolysis.
*6* The first step in the payoff phase is the oxidation of
glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate,
catalyzed by glyceraldehyde 3-phosphate dehydrogenase.
This is the first of the two energy-conserving reactions of
glycolysis that eventually lead to the formation of ATP.
The aldehyde group of glyceraldehyde 3-phosphate is oxidized,
not to a free carboxyl group but to a carboxylic acid anhydride
with phosphoric acid.
This type of anhydride, called an acyl phosphate, has a very
high standard free energy of hydrolysis.
So 1,3-Bisphosphoglycerate is an acyl phosphate.
Such compounds have a high phosphoryl-transfer potential; one
of its phosphoryl groups is transferred to ADP in the next step in
glycolysis.
The reaction is the sum of two processes: the oxidation of the
aldehyde to a carboxylic acid by NAD+ and the joining of the
carboxylic acid and orthophosphate to form the acyl-phosphate
product.
Much of the free energy of oxidation of the aldehyde group of
glyceraldehyde 3-phosphate is conserved by formation of the
acyl phosphate group at C-1 of 1,3-bisphosphoglycerate.
The acceptor of hydrogen in the glyceraldehyde 3-phosphate
dehydrogenase reaction is NAD+ , bound to a Rossmann fold.
The reduction of NAD+ proceeds by the enzymatic transfer of a
hydride ion (:H-) from the aldehyde group of glyceraldehyde 3-
phosphate to the nicotinamide ring of NAD+, yielding the
reduced coenzyme NADH. The other hydrogen atom of the
substrate molecule is released to the solution as H+ .
Glyceraldehyde 3-phosphate is covalently bound to the
dehydrogenase during the reaction. The aldehyde group of
glyceraldehyde 3-phosphate reacts with the --SH group of an
essential Cys residue in the active site, in a reaction analogous
to the formation of a hemiacetal, in this case producing a
thiohemiacetal. Reaction of the essential Cys residue with a
heavy metal such as Hg2+ irreversibly inhibits the enzyme.
IN OTHER WORDS
Let us consider the mechanism of glyceraldehyde 3-phosphate
dehydrogenase in detail.
In step 1, the aldehyde substrate reacts with the sulfhydryl
group of cysteine 149 on the enzyme to form a hemithioacetal.
Step 2 is the transfer of a hydride ion to a molecule of NAD +
that is tightly bound to the enzyme and is adjacent to the
cysteine residue. This reaction is favored by the deprotonation
of the hemithioacetal by histidine 176. The products of this
reaction are the reduced coenzyme NADH and a thioester
intermediate. This thioester intermediate has a free energy
close to that of the reactants.
In step 3, orthophosphate attacks the thioester to form 1,3-BPG
and free the cysteine residue. This displacement occurs only
after the NADH formed from the aldehyde oxidation has left the
enzyme and been replaced by a second NAD+. The positive
charge on the NAD+ may help polarize the thioester
intermediate to facilitate the attack by orthophosphate.
The favorable oxidation and unfavorable phosphorylation
reactions are coupled by the thioester intermediate, which
preserves much of the free energy released in the oxidation
reaction.
*7* The enzyme
phosphoglycerate kinase
transfers the high-energy
phosphoryl group from the
carboxyl group of 1,3-
bisphosphoglycerate to
ADP, forming ATP and
3-phosphoglycerate.
Notice that phosphoglycerate kinase is named for the reverse
reaction. Like all enzymes, it catalyzes the reaction in both
directions. This enzyme acts in the direction suggested by its
name during gluconeogenesis and during photosynthetic CO2
assimilation.
This Substrate-level phosphorylation (SLP) involves soluble
enzymes and chemical intermediates (1,3-bisphosphoglycerate
in this case).
The formation of ATP in this manner is referred to as substrate-
level phosphorylation because the phosphate donor, 1,3-BPG, is
a substrate with high phosphoryl-transfer potential.
Thus, the outcomes of the reactions catalyzed by
glyceraldehyde 3-phosphate dehydrogenase and
phosphoglycerate kinase are:
1. Glyceraldehyde 3-phosphate, an aldehyde, is oxidized to 3-
phosphoglycerate, a carboxylic acid.
2. NAD+ is concomitantly reduced to NADH.
3. ATP is formed from Pi and ADP at the expense of carbon
oxidation energy.
*8* The enzyme
phosphoglycerate mutase
catalyzes a reversible shift
of the phosphoryl group
between C-2 and C-3 of
glycerate; Mg2+ is
essential for this reaction.
Bisphosphoglycerate mutase catalyzes the conversion of 1,3-
bisphosphoglycerate to 2,3-bisphosphoglycerate, which is
converted to 3-phosphoglycerate by 2,3-bisphosphoglycerate
phosphatase (and possibly also phosphoglycerate mutase). This
alternative pathway involves no net yield of ATP from glycolysis.
However, it does serve to provide 2,3-bisphosphoglycerate,
which binds to hemoglobin, decreasing its affinity for oxygen
and so making oxygen more readily available to tissues.
In general, a mutase is an enzyme that catalyzes the
intramolecular shift of a chemical group, such as a phosphoryl
group.
This enzyme requires catalytic amounts of
2,3-bisphosphoglycerate to maintain an active-site histidine
residue in a phosphorylated form.
Examination of the first partial reaction reveals that the mutase
may function as a phosphatase it converts
2,3-bisphosphoglycerate into 2-phosphoglycerate. However, the
phosphoryl group remains linked to the enzyme. This
phosphoryl group is then transferred to 3-phosphoglycerate to
reform 2,3-bisphosphoglycerate.
*9* In the second glycolytic reaction that generates a
compound with high phosphoryl group transfer potential,
enolase promotes reversible removal of a molecule of water
(dehydration) from 2-phosphoglycerate to yield
phosphoenolpyruvate (PEP).
Although 2-phosphoglycerate and phosphoenolpyruvate contain
nearly the same total amount of energy, the loss of the water
molecule from 2-phosphoglycerate causes a redistribution of
energy within the molecule, greatly increasing the standard free
energy of hydrolysis of the phosphoryl group.
*10* The last step in glycolysis is
the transfer of the phosphoryl
group from phosphoenolpyruvate
to ADP, catalyzed by pyruvate
kinase, which requires K+ and
either Mg2+ or Mn2+ .
In this substrate-level phosphorylation, the product pyruvate
first appears in its enol form, then tautomerizes rapidly and
nonenzymatically to its keto form, which predominates at pH 7.
The overall reaction has a large, negative standard free energy
change, due in large part to the spontaneous conversion of the
enol form of pyruvate to the keto form.
The pyruvate kinase reaction is essentially irreversible under
intracellular conditions and is an important site of regulation.
Note that the energy released in the anaerobic conversion of
glucose into two molecules of pyruvate is -21 kcal mol-1
(-88 kJ mol-1).
In aerobic respiration, Electron transfer from NADH to O2 in
mitochondria provides the energy for synthesis of ATP by
respiration linked phosphorylation.
Glycolysis is tightly regulated in coordination with other energy-
yielding pathways to assure a steady supply of ATP. Hexokinase,
PFK-1, and pyruvate kinase are all subject to allosteric regulation
that controls the flow of carbon through the pathway and
maintains constant levels of metabolic intermediates.
The flux of glucose through the glycolytic pathway is regulated
to maintain nearly constant ATP levels (as well as adequate
supplies of glycolytic intermediates that serve biosynthetic
roles).
The required adjustment in the rate of glycolysis is achieved by
a complex interplay among ATP consumption, NADH
regeneration, and allosteric regulation of several glycolytic
enzymes—including hexokinase, PFK-1, and pyruvate kinase—
and by second-to-second fluctuations in the concentration of
key metabolites that reflect the cellular balance between ATP
production and consumption.
On a slightly longer time scale, glycolysis is regulated by the
hormones glucagon, epinephrine, and insulin, and by changes in
the expression of the genes for several glycolytic enzymes.
…MEDICAL CORRELATIONS…
Glucose uptake and glycolysis proceed about ten times faster in
most solid tumors than in noncancerous tissues. Tumor cells
commonly experience hypoxia (limited oxygen supply), because
they initially lack an extensive capillary network to supply the
tumor with oxygen. As a result, cancer cells more than 100 to
200 m from the nearest capillaries depend on anaerobic
glycolysis for much of their ATP production. They take up more
glucose than normal cells, converting it to pyruvate and then to
lactate as they recycle NADH.
The hypoxia-inducible transcription factor (HIF-1) is a protein
that acts at the level of mRNA synthesis to stimulate the
synthesis of at least eight of the glycolytic enzymes. This gives
the tumor cell the capacity to survive anaerobic conditions until
the supply of blood vessels has caught up with tumor growth.
2,3-bisphosphoglycerate binds to hemoglobin, decreasing its
affinity for oxygen and so making oxygen more readily available
to tissues.
Arsenite and mercuric ions react with the --SH groups of lipoic
acid and inhibit pyruvate dehydrogenase, as does a dietary
deficiency of thiamin, allowing pyruvate to accumulate.
Nutritionally deprived alcoholics are thiamin-deficient and may
develop potentially fatal pyruvic and lactic acidosis.
Patients with inherited pyruvate dehydrogenase deficiency,
which can be due to defects in one or more of the components
of the enzyme complex, also present with lactic acidosis,
particularly after a glucose load. Because of its dependence on
glucose as a fuel, brain is a prominent tissue where these
metabolic defects manifest themselves in neurologic
disturbances.
Deficiency of pyruvate kinase causes decreased production of
ATP from glycolysis. Red blood cells have insufficient ATP for
their membrane pumps, and a hemolytic anemia results,
although oxygen delivery to tissues is not necessarily affected.
As phosphoenolpyruvate accumulates, it is converted to 2-
phosphoglycerate, which leads to increased levels of 2,3-
bisphosphoglycerate in the red blood cells. The elevated levels
of 2,3-bisphosphoglycerate promote oxygen release from
hemoglobin in the tissues to an extent that is greater than in the
presence of normal 2,3-bisphosphoglycerate levels.
Inherited aldolase A deficiency and pyruvate kinase deficiency in
erythrocytes cause hemolytic anemia. The exercise capacity of
patients with muscle phosphofructokinase deficiency is low,
particularly on high-carbohydrate diets. By providing an
alternative lipid fuel, e.g., during starvation, when blood free
fatty acids and ketone bodies are increased, work capacity is
improved.
GLUT1 deficiency can have serious consequences. The GLUT1
transporter translocates glucose across the blood–brain barrier.
When one allele is defective, the rate of glucose entry into the
nervous system is insufficient for the cells’ needs, leading to
seizures, developmental delays, and microcephaly. The
treatment consists of a ketogenic diet, one high in fat, in order
to produce ketone bodies as an alternative energy source for
the nervous system.
An increase of lactate levels in the blood causes an acidosis
(lactic acidosis). This condition can result from hypoxia or
alcohol ingestion. Lack of oxygen slows down the electron
transport chain, resulting in increased NADH levels. High NADH
levels cause more than normal amounts of pyruvate to be
converted to lactate. High NADH levels from alcohol metabolism
also cause increased conversion of pyruvate to lactate. Thiamine
deficiency, which is common in alcoholics, decreases pyruvate
dehydrogenase activity, causing pyruvate to accumulate and
form lactate. Thiamine deficiency also slows down the TCA cycle
at the α-ketoglutarate dehydrogenase step. This and other
conditions that slow down the TCA cycle can also produce a
lactic acidosis.
Credit goes to----.
Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme
Usage subject to terms and conditions of license.
All right preserved.
Harper’s Illustrated Biochemistry, Twenty-Sixth Edition
LUBERT STRYER Biochemistry, Fifth Edition
Lehninger principles of biochemistry 4th ED.
BRS Biochemistry, Molecular Biology, and Genetics 6TH ED.
SUMMARIZED BY// ALY BARAKAT