Reaction 7: 1,3bisphosphoglycerate to 3phosphoglycerate

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Reaction 7 1,3-bisphosphoglycerate to
3-phosphoglycerate
phosphoryl group from the acyl phosphate of
1,3-BG is transferred to ADP by phosphoglycerate
kinase This is a substrate level
phosphorylation reaction
terminial phosphoryl oxygen of ADP makes a
nucleophilic attack on the C-1 phosphorous of 1,3
BPG because of the high phosphate group transfer
potential of 1,3BPG - it has a strong tendency to
transfer its acyl-phosphate group to ADP with the
resulting formation of ATP
Q What is the net yeild of ATP at this stage of
gycolysis? A Zero Two ATPs per mole of glucose
were invested to generate 2 moles of triose
phosphate This reaction generates one ATP from
each mole of triose phosphate or two ATPs per
mole of glucose
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Reaction 8 3-phosphoglycerate to 2-
phosphoglycerate
this is an isomerization reaction catalyzed by
phosphoglycerate mutase in which the phosphate at
position 3 is transferred to postion 2. Mg2 is
required
Even though the reaction is slightly endergonic
understandard conditions, the intra cellular
level of 3-phosphoglycerate is high relative to
of 2-phosphoglycerate (2PG) so that in vivo the
reaction proceeds to the right
This is not a simple intra molecular phosphoryl
transfer Active form of the enzyme has a
phosphohistidine residue at the active site It
adds a phosphate to C-2 and removes the phosphate
from C-3
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Reaction 9 2- phosphoglycerate to
phosphoenolpyruvate
an enol is formed by the dehydration of 2PG in a
reaction catalzyed by enolase
the effect of this reaction is to increase the
free energy of hydrolysis of the phosphate bond
from -15.6 kJ/mol for 2PG to -61.9 kJ/mol for
phosphoenolpyruvate ( increase the transfer
potential of the phosphoryl group) The loss of
the water molecule from 2-PG causes a
redistribution of energy within the molecule,
greatly increasing the standard free energy of
hydrolysis of the phosphate group
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Reaction 10 phosphoenolpyruvate to pyruvate
This is a substrate level phosphorylation in
which PEP transfers its phosphate to ADP in a
reaction catalyzed by pyruvate kinase (the enzyme
requires Mg2 and K) ATP is concomitantly
generated.
REACTION ATP change per
glucose glucose glucose 6-phosphate -1 fruct
ose 6-phosphate fructose 1,6 bisphosphate -1 (2
) 1,3-bisphosphoglycerate (2) 3-phosphoglycerate
2 (2) phosphoenolpyruvate (2)
pyruvate 2 NET 2
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Pyruvate Kinase Deficiency and hemolytic anemia

• Mature erythrocytes are absolutely dependent on
  glycolysis for ATP production
• Without ATP the ion pumps that maintain the
  concave shape of erythrocytes (a characteristic
  that helps them slip through capillaries as they
  deliver oxygen to tissue cant function properly
  and the erythrocyte swell and burst)
• This results in hemolytic anemia (anemia due to
  excessive erythrocyte destruction)

Pyruvate kinase deficiency is the most common
genetic defect of glycolysis Pyruvate Kinase
deficient patients have 5-25 the normal levels
of the enzyme in RBCs
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Intermediates in glycolysis are used in many
other metabolic pathways and are the building
blocks of a variety of important compounds
Is molecular oxygen required for glycolysis?
Glycolysis was used by organisms _at_ 1 billion
years before photosynthetic organisms began
contributing O2 to the earths atmosphere (_at_2.5
billion years ago) as a way to generate
energy today, in aerobic organisms 1. under
aerobic conditions pyruvate enters mitochondria
and is completely oxidized to CO2 and H2O 2.
under anerobic conditions pyruvate is reduced to
lactate or ethanol
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Entry of other saccarides into glycolysis
lactose intolerance a result of a deficiency of
lactase, the enzyme that cleaves lactose into
glucose and galactose Since there is no
mechanism for uptake of dissacharides, lactose
accumulates in the small intestine and the large
osmotic effect of the unabsorbed lactose leads to
influx of fluid into the small intestine Percenta
ge of adults who are lactase difficient varies
among populations example over 80 of Southeast
Asians are lastase deficient less than 10 of
Northern Europeans are lastase deficient
Galactosemia a variety of genetic disorders
that involve a failure to metabolize galactose so
that it (or one of its metabolites) accumulates
in blood and tissues
Fructose is present as the free sugar in many
fruits as well as being part of sucrose two
pathways for the metabolism of fructose (next
page)
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Regulation of glycolysis

• The glycolytic pathway has a duel role
• it degrades glucose to generate ATP and
• it provides building blocks for synthetic
  reactions
• the rate of conversion of glucose into pyruvate
  is regulated to meet both needs
• In metabolic pathways, enzymes catalyzing
  irreversibe reactions are potential sites of
  control
• In glycolysis, reactions catalyzed by the
  following enzymes are essentially irreversible
• Hexokinase
• Phosphofructokinase
• pyruvate kinase
• Their activities are regulated by
• the reversible binding of allosteric effectors
  (milliseconds)
• covalent modification (seconds)
• transciptional control to vary the amount of
  enzyme present (hours)

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Metabolic fates of pyruvate
While the sequence of reactions from glucose to
pyruvate is similar in all organisms and in all
kinds of cells, the fate of pyruvate is
variable the fate of pyruvate depends on the
oxidation state of the cell
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Formation of lactate from pyruvate
The reduction of pyruvate by NADH to form lactate
is catalyzed by lactate dehydrogenase Lactate is
formed in a number of microorganisms normally and
in cells of higher organisms when the amount of
oxygen is limiting (as in muscle during intense
activity)
Remember that in the glyceraldehyde 3 phosphate
dehydrogenase reaction NAD is reduced to NADH
and this NADH is then REPLACED with another
molecule of NAD (to regenerate the enzyme active
site) The NADH is used by lactate dehydrogenase
and the NAD generated in used in the G3PD
reaction to displace the NADH
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Isozymes provide a means of regulation specific
to distinct tissues and developmental stages -
Isozymes of LDH
H4 has higher affinity for substrate than M4 H4
is allosterically inhibited by high levels of
pyruvate, M4 is not
H4 is designed to oxidize lactate to pyruvate,
which is then used by the heart through aerobic
metabolism (heart muscle never functions
anaerobically) M4 is optimized to function in
the reverse direction from H4 (M4 is optimized
to convert pyruvate to lactate so that glycolysis
can proceed under anaerobic conditions)
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What happens to the lactate produced by muscle
cells?
Cori cycle
What happens to lactate produced by
microorganisms?
Many microorganisms ferment glucose and other
hexoses to lactate Certain lactobacilli and
streptococci, for example, ferment the lactose in
milk to lactic acid The dissociation of lactic
acid to lactate and H in the fermentation
mixture lowers the pH, denaturing casein and
other milk proteins and causing them to
precipitate Under the correct, controlled
conditions, the curdling process produces cheese
or yogurt, depending on the microorganism
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Carbohydrate catabolism
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Formation of ethanol from pyruvate
yeast and several other microorganisms ferment
pyruvate to ethanol and CO2 (instead of to
lactate) in a two step proccess This first step,
the (nonoxidative) decarboxylation of pyruvate is
catalyzed by pyruvate decarboxylase Pyruvate
decarboxylase is present in brewers yeast and
bakers yeast and in all other organisms in which
alcohol fermentation occurs (including some
plants) The CO2 released is responsible for
dough to rise and for champagne to bubble But
whats the mechanism?
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TPP (the coenzyme form of vitamin B1) plays an
important role in the cleavage of bonds adjacent
to a a carbonyl group (such as the
decarboxylation of a keto acids) and in
chemical rearrangements in which an activated
aldehyde group is transferred from one carbon
atom to another
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Loss of the proton at C-2 creates a carbanion
Acetaldehyde is released
The TPP carbanion acts as a nucleophile and
attacks the carbonyl group of pyruvate
The carbanion is protonated to form hydroxyethyl
TPP
Decarboxylation produces a carbanion that is
stabilized by the thiazolium ring
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Formation of ethanol from pyruvate - continued
The active site contains a zinc residue bound to
two cysteine residues and one histidine
residue The zinc ion binds the acetaldehyde
substrate through its oxygen atom, polarizing it
so that it more easily accepts a hydride (light
blue) from NADH
The nicatinamide binding half (yellow) is
structurally similar to the adenine-binding half
(red) The two halves together form a structural
motif called a Rossmann fold The NAD molecule
binds in an extended conformation
NAD generated in the reduction of acetaldehyde
to ethanol is consumed in the oxidation of
glyderaldehyde 3- phosphate
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NAD generated in the reduction of acetaldehyde
to ethanol (and lactate) is consumed in the
oxidation of glyderaldehyde 3- phosphate
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Animal tissue also contains alcohol
dehydrogenase ( even though ethanol is not a
major metabolic product in animal cells)

• If ethanol is present it will be oxidized by this
  enzyme in the liver
• consequences are
• massive reduction of NAD to NADH H so the
  bodies redox state is altered
• NAD is not available for the many reactions
  that require it so there is consequent inhibition
  of energy generation
• acetaldehyde is toxic and cause some of the
  symptoms of a hangover

• How the body utilizes alcohol
• alchohol is not digested and is quickly
  absorbed
• _at_20 is absorbed directly across the walls of an
  empty stomach and can reach
• the brain within one minute
• with a full stomach, this effect is delayed
  because alcohol absorption is slowed
• (there is alchohol dehydrogenase in the stomach
  which also reduces the amount of alcohol reaching
  the blood)
• alcohol is also absorbed in the duodenum and
  from here on it gets absorbed and metabolized
  before most nutrients
• liver cells prefer fatty acids as their fuel,
  but when alcohol is present, liver cells are
  forced to metabolize alcohol and let the fatty
  acids accumulate

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Glucose Catabolism in Cancerous Tissue
Glucose uptake and glycolysis proceed about ten
times faster in most solid tumors than in
noncancerous tissues why? 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 mm from a capillary 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 high glycolytic rate may
also result in part form smaller numbers of
mitochondria in tumor cells and less ATP from
mitochondrial oxidative phosphorylation means
that more is needed from glycolyis In addition,
some tumor cells overproduce several glycolytic
enzymes including an isozyme of hexokinase that
is insensitive to feedback inhibition by
glucose-6-phosphate
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Anaerobic glycolysis Making beer
Start with cereal grains (barley) which contain
the polysaccharides amylose and
amylopectin) initiate a process called malting
in which the grains are allowed to germinate till
they hydrolytic enzymes required for
polysaccharide breakdown are made
Process is stopped by controlled heating and
produces malt Wort is prepared from the
malt, by mixing the malt with water and mashing
or crushing the malt so that the hydrolytic
enzymes formed by malting degrade the
polysaccharides into monosaccharides The liquid
wort is separated from the debris and boiled with
hops to give flavor - then cooled
Yeast cells are added at this stage Initially,
because oxygen is present, the yeast cells
convert pyruvate into acetyl CoA and continue
through the citric acid cycle and respiration and
are multiplying When oxygen is consumed, yeast
switch to anaerobic fermentation and produce
ethanol from pyruvate
Final stage is controlling the amount of foam
produced Foam is produced from dissolved
proteins if too much catalytic activity
goes on in the earlier steps, not enough
undegraded proteins flat beer If not
enough catalytic activity too many undegraded
proteins cloudy beer
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Microbial fermentations yield other end products
of commercial value
1910 Chaim Weizmann (who later became the first
president of Israel) discovered that the bacteria
Clostridium acetobutyricium ferments starch to
butanol and acetone
Now readily available carbohydrate rich material
such as corn starch or molasses are fermented in
huge industrial size vats by the appropriate
microorganism into methanol glycerol ethanol
isopropanol butanol various acids such
as formic, acetic, proprionic,
butyric succinic
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