Glycolysis

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The fates of the glucose

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Overview

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Reaction 1: Activation of the
glucose
The first reaction consumes
ATP. This phosphate makes
the glucose a negatively
charged molecule so that it
stays inside the cell and
prevents it from leaving
through channels.

Kinases are enzymes that

transfer ions from one
molecule to another. The
hexokinase is an enzyme that
transfers the phosphate from
the ATP to a hexose (in this
case the glucose).
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Reaction 1: Activation of the
glucose

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Reaction 2: Rearrangement of the
glucose
The second reaction rearranges
the six carbons of the glucose-6-
phosphate. Glucose-6-phosphate
is an aldose, a sugar with an
aldehyde group (in C1). The
phosphoglucose isomerase
transforms the aldehyde group
into a keto group. Fructose 6-
phosphate is a ketose, a sugar
with a keto group (in C2).

Because these two are isomers,

this is an isomerisation reaction
(catalysed by an isomerase).

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Reaction 3: Phosphorylation of
the fructose
The third reaction consumes a
second ATP and is catalysed by
phosphofructokinase (PFK). The
kinase transfers the phosphoryl group
from the ATP to the C1 of the fructose
6-phosphate.

PFK is inhibited by ATP via allosteric

inhibition. The active site of the PFK
recognises ATP and the reaction with
the fructose takes places. In a
different part of the enzyme, ATP can
also bind and change the
configuration of the active site, so that
ATP cannot fit in. The inhibition site
has higher affinity to ATP than the
active site (making glycolysis tightly
regulated). 27
Reaction 4: Splitting of the
fructose 1,6-bisphosphate
The fourth reaction is the breaking down of
the fructose 1,6-bisphosphate into two
trioses. This reaction is catalysed by the
aldolase, an enzyme that breaks the bond
between C3 and C4 in the fructose. The
products are two different trioses: an aldose
(glyceraldehyde 3-phosphate), and a ketose
(dihydroxyacetone phosphate).

The aldolase is a very specific enzyme and it

differs in its affinity to substrates depending
of the organ: aldolase A is expressed in the
muscles and brain, aldolase B in the liver,
kidney and enterocytes, and aldolase C in
the brain. In humans, they are encoded in
different chromosomes (16, 9 and 17,
respectively). 28
Reaction 5: Second isomerisation

To move forward, the triose needs to be an

aldose. The triose phosphate isomerase
(TPI) catalyses this reaction by rearranging
the functional groups of the
dihydroxyacetone into the glyceraldehyde 3-
phosphate.

The TPI catalyses this reaction a billion times

faster than the rate at which the
isomerisation occurs on its own in solution. It
is one of the few enzymes to be catalytically
perfect, because it is limited only by the rate
of diffusion of the substrates into the active
site. The high speed of this enzyme makes
the glycolysis more synchronous.
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 Reaction 6: Oxidation of the
trioses
1 mol of glucose produces a yield of 2
moles of 1,3 biphosphate glycerate, 2
moles of NADH + H+ and 4 moles of
electrons (reaction 6 occurs twice per
glucose).
The sixth reaction is a dehydrogenation,
meaning that one hydrogen is removed from
the molecule. This is an oxidation reaction, but
dehydrogenases can oxidise and reduce
depending on the substrates.
This hydrogen, with one electron, is transferred
from the glyceraldehyde 3-phosphate to a
molecule of NAD + by the glyceraldehyde
phosphate dehydrogenase to produce NADH.
A second electron gets removed from the
oxygen and an inorganic phosphate is added
to the triose. This produces NADH + H+ and
1,3-bisphosphate glycerate.
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Reaction 7: Restauration of the
ATP
In the seventh reaction, a phosphoglycerate
kinase (PGK) removes a phosphoryl group
from the 1,3-biphosphoglycerate to a
molecule of adenosine diphosphate (ADP) to
produce ATP and 3-phosphoglycerate.

Each molecule of glucose produces two

trioses and, by reaction 7, the ATP
consumed in the beginning of the glycolysis
(reactions 1 and 3) is fully restored.

In humans, PGK has two variants: PGK-1

encoded in the X-chromosome, and PGK-2,
encoded in chromosome 6 and only
expressed by spermatozoids that do not
carry chromosome X. 31
Reaction 8: Rearrangement of the
trioses
The eighth reaction is also an isomerisation
reaction, but instead of changing the
functional group, the phosphoglycerate
mutase (PGM) moves the phosphoryl from
C3 to C2, producing 2-phosphoglycerate.
Mutases move a functional group without
changing the overall structure.

This enzyme is formed by two subunits. One subunit originates in the muscle
tissue (subunit m), and the other one in all types of tissues (subunit b, since it was
isolated first in the brain). Smooth muscle has the enzyme with two subunits m
(mm-PGM), cardiac and skeletal muscle have subunits m and b (mb-PGM), and
the rest of the tissues have two enzymes with two subunits b (bb-PGM).

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Reaction 9: Hydrolysis in the
trioses
The ninth reaction is the liberation of water
from 2-phosphoglycerate by the enzyme
enolase, producing water and
phosphoenolpyruvate.

Enolase is formed by three subunits and

each subunit is encoded in a different gene.
The subunits are named α, β, and γ. Enolase
1 has the structure αα and is found in all
normal human cells, more common in the
liver, brain, spleen, kidneys and the adipose
tissue. Enolase 3 (ββ) is restricted to the
muscle. Enolase 2 (γγ) is highly produced in
the neurones and other nervous tissue.

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Reaction 10: ATP production
The tenth and final reaction is the stage
where glycolysis produces energy, by
transferring the phosphoryl group from the
phosphoenolpyruvate to ADP producing
pyruvate, an acid. The pyruvate kinase
(PEP) was named before discovering that
under physiological conditions PEP cannot
transfer the phosphoryl from pyruvate.

PEP is inactive in the cytosol until a fructose

1,6-bisphosphate binds the allosteric site.
Just like haemoglobin, PEP has two states R
and T. State R has high affinity for the
substrates and is stabilised by
phosphoenolpyruvate and fructose 1,6-
biphosphate. State T has low affinity for the
substrates and is stabilised by ATP. State T
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is the inactive state.
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 Summary
• If NADH cannot be oxidised through aerobic
respiration, another electron acceptor is used.
• Most organisms will use some form of
fermentation to accomplish the regeneration of
NAD+, ensuring the continuation of glycolysis.
• The regeneration of NAD+ in fermentation is
not accompanied by ATP production; therefore,
the potential of NADH to produce ATP using an
electron transport chain is not utilised.
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 Summary
• Glycolysis is the first pathway used in the
breakdown of glucose to extract energy. It was
probably one of the earliest metabolic
pathways to evolve and is used by nearly all of
the organisms on earth.
• Glycolysis consists of two parts:
• The first part prepares the six-carbon ring of
glucose for cleavage into two three-carbon
sugars. ATP is invested in the process during
this half to energise the separation.
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 Summary
• The second half of glycolysis extracts ATP
and high-energy electrons from hydrogen
atoms and attaches them to NAD+.
• Two ATP molecules are invested in the first
half and four ATP molecules are formed by
substrate phosphorylation during the
second half.
• This produces a net gain of two ATP and
two NADH molecules for the cell.
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