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 GLYCOLYSIS

 Glycolysis is one of the principle

pathways for generating ATP in cells and
is present in all cell types.
 The central role of glycolysis in fuel
metabolism is related to its ability to
generate ATP with, and without, oxygen.
The oxidation of glucose to pyruvate
generates ATP from substrate-level
phosphorylation (the transfer of phosphate
from high-energy intermediates of the
pathway to ADP) and NADH.
Subsequently, the pyruvate may be
oxidized to CO2 in the TCA cycle and ATP
generated from electron transfer to
oxygen in oxidative phosphorylation.
If the pyruvate and NADH from glycolysis
are converted to lactate (anaerobic
glycolysis), ATP can be generated in the
absence of oxygen, via substrate-level
phosphorylation.
 The Reactions of Glycolysis

 The glycolytic pathway, which cleaves 1

mole of glucose to 2 moles of the 3-carbon
compound pyruvate, consists of a preparative
phase and an ATP-generating phase.
 In the initial preparative phase of glycolysis,
glucose is phosphorylated twice by ATP and
cleaved into two triose phosphates.
The ATP expenditure in the beginning of the
preparative phase is sometimes called
“priming the pump,” because this initial
utilization of 2 moles of ATP/mole of
glucose results in the production of 4
moles of ATP/mole of glucose in the ATP-
generating Phase.
In the ATP-generating phase,
glyceraldehyde 3-phosphate (a triose
phosphate) is oxidized by NAD and
phosphorylated using inorganic
phosphate.
The high energy phosphate bond
generated in this step is transferred to
ADP to form ATP.
 The remaining phosphate is also
rearranged to form another high-energy
phosphate bond that is transferred to
ADP.
There were 2 moles of triose
phosphate formed, the yield from the
ATP-generating phase is 4 ATP and 2
NADH.
The result is a net yield of 2 moles of
ATP, 2 moles of NADH, and 2 moles of
pyruvate per mole of glucose.
 CONVERSION OF GLUCOSE TO GLUCOSE 6-
PHOSPHATE

Glucose metabolism begins with

transfer of a phosphate from ATP to
glucose to form glucose-6-P.
 Phosphorylation of glucose commits it
to metabolism within the cell because
glucose-6-P cannot be transported back
across the plasma membrane.
Glucose-6-P is a branchpoint in carbohydrate
metabolism.
 It is a precursor for almost every pathway that
uses glucose, including glycolysis, the pentose
phosphate pathway, and glycogen synthesis.
 From the opposite point of view, it also can be
generated from other pathways of carbohydrate
metabolism, such as glycogenolysis (breakdown
of glycogen), the pentose phosphate pathway,
and gluconeogenesis.
 Hexokinases, the enzymes that
catalyze the phosphorylation of glucose,
are a family of tissue-specific isoenzymes
that differ in their kinetic properties.
 The isoenzyme found in liver and β
cells of the pancreas has a much higher
Km than other hexokinases and is called
glucokinase.
 CONVERSION OF GLUCOSE-6-P TO THE TRIOSE
PHOSPHATES

In the remainder of the preparative phase of

glycolysis, glucose-6-P is isomerized to
fructose 6-phosphate (fructose-6-P), again
phosphorylated, and subsequently cleaved
into two 3-carbon fragments.
 The isomerization, which positions a keto
group next to carbon 3, is essential for the
subsequent cleavage of the bond between
carbons 3 and 4.
The next step of glycolysis, phosphorylation
of fructose-6-P to fructose 1,6- bisphosphate
(fructose-1,6-bisP) by phosphofructokinase-1
(PFK-1), is generally considered the first
committed step of the pathway.
 This phosphorylation requires ATP and is
thermodynamically and kinetically irreversible.
PFK-1 irrevocably commits glucose to the
glycolytic pathway.
 PFK-1 is a regulated enzyme in cells, and its
regulation controls the entry of glucose into
glycolysis.
Fructose-1,6-bisP is cleaved into two
phosphorylated 3-carbon compounds (triose
phosphates) by aldolase.
 Dihydroxyacetone phosphate (DHAP) is
isomerized to glyceraldehyde 3-phosphate
(glyceraldehyde-3-P), which is a triose phosphate.
 Thus, for every mole of glucose entering
glycolysis, 2 moles of glyceraldehyde-3-P continue
through the pathway.
 OXIDATION AND SUBSTRATE LEVEL
PHOSPHORYLATION

In the next part of the glycolytic pathway,

glyceraldehyde-3-P is oxidized and
phosphorylated so that subsequent
intermediates of glycolysis can donate
phosphate to ADP to generate ATP.
The first reaction in this sequence, catalyzed by
glyceraldehyde- 3-P dehydrogenase, is really the
key to the pathway.
 This enzyme oxidizes the aldehyde group of
glyceraldehyde-3-P to an enzyme-bound carboxyl
group and transfers the electrons to NAD to form
NADH.
The oxidation step is dependent on a cysteine
residue at the active site of the enzyme, which
forms a high-energy thioester bond during the
course of the reaction.
To transfer the remaining low-energy
phosphoester on 3-phosphoglycerate to ADP, it
must be converted into a high-energy bond.
 This conversion is accomplished by moving
the phosphate to the second carbon (forming 2-
phosphoglycerate) and then removing water to
form phosphoenolpyruvate (PEP).
 SUMMARY OF THE GLYCOLYTIC PATHWAY

The overall net reaction in the glycolytic pathway

is:
 Oxidative Fates of Pyruvate and NADH

The NADH produced from glycolysis

must be continuously reoxidized back to
NAD to provide an electron acceptor for
the glyceraldehyde-3-P dehydrogenase
reaction and prevent product inhibition.
 Without oxidation of this NADH,
glycolysis cannot continue.
There are two alternate routes for oxidation
of cytosolic NADH.
One route is aerobic, involving shuttles that
transfer reducing equivalents across the
mitochondrial membrane and ultimately to
the electron transport chain and oxygen.
The other route is anaerobic (without the use of
oxygen).
 In anaerobic glycolysis, NADH is reoxidized in the
cytosol by lactate dehydrogenase, which reduces
pyruvate to lactate.
The fate of pyruvate depends on the route
used for NADH oxidation.
 If NADH is reoxidized in a shuttle system,
pyruvate can be used for other pathways, one
of which is oxidation to acetyl-CoA and entry
into the TCA cycle for complete oxidation.
Alternatively, in anaerobic glycolysis,
pyruvate is reduced to lactate and diverted
away from other potential pathways.
The use of the shuttle systems allows
for more ATP to be generated than by
anaerobic glycolysis by both oxidizing
the cytoplasmically derived NADH in the
electron transport chain and by allowing
pyruvate to be oxidized completely to
CO2.
 Anaerobic Glycolysis

When the oxidative capacity of a cell is limited

(e.g., the red blood cell, which has no mitochondria),
the pyruvate and NADH produced from glycolysis
cannot be oxidized aerobically.
 The NADH is therefore oxidized to NAD in the
cytosol by reduction of pyruvate to lactate.
 This reaction is catalyzed by lactate
dehydrogenase (LDH)
The net reaction for anaerobic glycolysis is:
ENERGY YIELD OF AEROBIC VERSUS ANAEROBIC
GLYCOLYSIS

In both aerobic and anaerobic glycolysis,

each mole of glucose generates 2 moles of
ATP, 2 of NADH and 2 of pyruvate.
The energy yield from anaerobic glycolysis
(glucose to 2 lactate) is only 2 moles of ATP
per mole of glucose, as the NADH is recycled
to NAD by reducing pyruvate to lactate.
 Neither the NADH nor pyruvate produced is
thus used for further energy generation.
When oxygen is available, and cytosolic
NADH can be oxidized via a shuttle system,
pyruvate can also enter the mitochondria and
be completely oxidized to CO2 via PDH and the
TCA cycle.
 The oxidation of pyruvate via this route
generates roughly 12.5 moles of ATP per mole
of pyruvate.
If the cytosolic NADH is oxidized by the
glycerol 3-P shuttle, approximately 1.5
moles of ATP are produced per NADH.
 If, instead, the NADH is oxidized by the
malate–aspartate shuttle, approximately
2.5 moles are produced.
The two NADH molecules produced during
glycolysis can lead to 3 to 5 molecules of ATP
being produced, depending on which shuttle
system is used to transfer the reducing
equivalents.
Each pyruvate produced can give rise to
12.5 molecules of ATP, altogether 30 to 32
molecules of ATP can be produced from one
mole of glucose oxidized to carbon dioxide.
ACID PRODUCTION IN ANAEROBIC GLYCOLYSIS

If the amount of lactate generated exceeds

the buffering capacity of the blood, the pH
drops below the normal range, resulting in
lacticacidosis.
 TISSUES DEPENDENT ON ANAEROBIC GLYCOLYSIS

Many tissues, including

• red and white blood cells,
• the kidney medulla,
•the tissues of the eye,
•and skeletal muscles,
rely on anaerobic glycolysis for at least a
portion of their ATP requirements.
 Tissues (or cells) that are heavily dependent
on anaerobic glycolysis usually have a
•Low ATP demand,
•High levels of glycolytic enzymes,
•And few capillaries, such that oxygen must diffuse over a
greater distance to reach target cells.
 The lack of mitochondria, or the increased
rate of glycolysis, is often related to some
aspect of cell function.
 FATE OF LACTATE

Lactate released from cells undergoing anaerobic

glycolysis is taken up by other tissues (primarily the
liver, heart, and skeletal muscle) and oxidized back
to pyruvate.
 In the liver, the pyruvate is used to synthesize
glucose (gluconeogenesis), which is returned to the
blood.
The cycling of lactate and glucose between
peripheral tissues and liver is called the Cori cycle
 OTHER FUNCTIONS OF GLYCOLYSIS

Intermediates of the pathway can be converted to

•Ribose 5-phosphate, the sugar incorporated into
nucleotides such as ATP.
•UDP-glucose,
•Mannose,
•And sialic acid, are also formed from intermediates of
glycolysis.
•Serine is synthesized from 3-phosphoglycerate, and
alanine from pyruvate.
•The backbone of triacylglycerols, glycerol 3-phosphate, is
derived from dihydroxyacetone phosphate in the
glycolytic pathway.
•The liver synthesizes fatty acids from the
pyruvate generated by glycolysis.
• It also synthesizes glucose from lactate,
glycerol 3-phosphate, and amino acids in the
gluconeogenic pathway, which is principally a
reversal of glycolysis.
REGULATION OF GLYCOLYSIS BY THE NEED
FOR ATP

• When ATP is needed, glycolysis is activated

• When ATP levels are sufficient, glycolysis activity decreases

Control points
1. Hexokinase
2. PFK-1
3. Pyruvate kinase
REGULATION OF GLYCOLYSIS BY THE NEED
FOR ATP

One of the major functions of glycolysis is the

generation of ATP, and, therefore, the pathway
is regulated to maintain ATP homeostasis in all
cells.
 Phosphofructokinase- 1 (PFK-1) and pyruvate
dehydrogenase (PDH), which links glycolysis and
the TCA cycle, are both major regulatory sites
that respond to feedback indicators of the rate
of ATP utilization.
Relationship between ATP, ADP, and AMP Concentrations

The AMP levels within the cytosol provide a

better indicator of the rate of ATP utilization
than the ATP concentration itself.
 Regulation of Hexokinases

Hexokinases exist as tissue-specific

isoenzymes whose regulatory properties reflect
the role of glycolysis in different tissues.
 In most tissues, hexokinase is a low-Km
enzyme with a high affinity for glucose.
It is inhibited by physiologic concentrations of
its product, glucose-6-P.
If glucose-6-P does not enter glycolysis or
another pathway, it accumulates and decreases
the activity of hexokinase.
In the liver, the isoenzyme glucokinase
is a high-Km enzyme that is not readily
inhibited by glucose-6-P.
Thus, glycolysis can continue in liver
even when energy levels are high so that
anabolic pathways, such as the synthesis
of the major energy storage compounds,
glycogen and fatty acids, can occur.
 Regulation of PFK-1

Phosphofructokinase-1 (PFK-1) is the

rate-limiting enzyme of glycolysis and
controls the rate of glucose-6-P entry into
glycolysis in most tissues.
 PFK-1 is an allosteric enzyme that has a
total of six binding sites:
•two are for substrates (Mg-ATP and
fructose- 6-P)
• and four are allosteric regulatory sites.
The allosteric sites for PFK-1 include
•an inhibitory site for MgATP,
•an inhibitory site for citrate and other anions,
•an allosteric activation site for AMP,
•and an allosteric activation site for fructose 2,6-
bisphosphate (fructose-2,6-bisP) and other
bisphosphates.
ALLOSTERIC REGULATION OF PFK-1 BY AMP AND ATP

ATP binds to two different sites on

the enzyme, the substrate binding site
and an allosteric inhibitory site.
 Under physiologic conditions in the
cell, the ATP concentration is usually
high enough to saturate the substrate
binding site and inhibit the enzyme by
binding to the ATP allosteric site.
This effect of ATP is opposed by AMP,
which binds to a separate allosteric activator
site).
 For most of the PFK-1 isoenzymes, the
binding of AMP increases the affinity of the
enzyme for fructose 6-P.
 REGULATION OF PFK-1 BY FRUCTOSE 2,6-BISPHOSPHATE

Fructose-2,6-bisP is also an allosteric activator of PFK-1

that opposes the ATP inhibition.
 It is a bifunctional enzyme with two separate domains, a
kinase domain and a phosphatase domain.
At the kinase domain, fructose-6-P is phosphorylated to
fructose-2,6-bisP and at the phosphatase domain,
fructose-2,6-bisP is hydrolyzed back to fructose-6-P. PFK-2
is regulated through changes in the ratio of activity of the
two domains.
For example, in skeletal muscles, high concentrations of
fructose-6-P activate the kinase and inhibit the
phosphatase, thereby increasing the concentration of
fructose-2,6-bisP and activating glycolysis.
 ALLOSTERIC INHIBITION OF PFK-1 AT THE CITRATE SITE

The function of the citrate–anion allosteric site is to integrate glycolysis

with other pathways. For example, the inhibition of PFK-1 by citrate may
play a role in decreasing glycolytic flux in the heart during the oxidation
of fatty acids.
Pyruvate Dehydrogenase Regulation and Glycolysis

When tissues do not have an adequate

supply of O2 to meet their ATP demands, the
increased NADH/NAD ratio inhibits pyruvate
dehydrogenase, but AMP activates glycolysis.
A proportion of the pyruvate will then be
reduced to lactate to allow glycolysis to
continue.
 LACTIC ACIDEMIA

Lactic acidosis generally results from a

greatly increased NADH/NAD ratio in tissues.
 The increased NADH concentration prevents
pyruvate oxidation in the TCA cycle and directs
pyruvate to lactate.
 To compensate for the decreased ATP
production from oxidative metabolism, PFK-1,
and, therefore, the entire glycolytic pathway is
activated.
For example, consumption of high
amounts of alcohol, which is rapidly
oxidized in the liver and increases NADH
levels, can result in a lactic acidosis.
 Hypoxia in any tissue increases lactate
production as cells attempt to
compensate for a lack of O2 for
oxidative phosphorylation.
A number of other problems that interfere either with
the electron transport chain or pyruvate oxidation in the
TCA cycle result in lactic acidemia. For example, OXPHOS
diseases (inherited deficiencies in subunits of complexes
in the electron transport chain, such as MERFF) increase
the NADH/NAD ratio and inhibit PDH.
Impaired PDH activity from an inherited deficiency of E1
(the decarboxylase subunit of the complex), or from
severe thiamine deficiency, increases blood lactate levels .
Pyruvate carboxylase deficiency also can result in lactic
acidosis, because of an accumulation of pyruvate
Lactic acidosis can also result from inhibition of
lactate utilization in gluconeogenesis (e.g., hereditary
fructose intolerance, which is due to a defective
aldolase gene).
If other pathways that use glucose-6-P are blocked,
glucose-6-P can be shunted into glycolysis and lactate
production (e.g., glucose 6-phosphatase deficiency).
 Assignment One

Summarize Glycolysis in one

diagram