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GLYCOLYSIS
Before glycolysis
• Before we get to this step, carbohydrates such as starch
are broken down into glucose via -amylase
Glycolysis in β Cells
• Glucose in the blood
•
•
•
•
enters  cells of the
pancreas through GLUT2
transporters (KM = 15-20
mM)
The  cell metabolizes
glucose to pyruvate
generating ATP
Increase in ATP/ADP ratio
closes K+ channel
This then opens a Ca2+
channel ultimately
transporting insulin out of
the cell
Energy change results in
insulin secretion
GLUT2 transporter
Before Glycolysis
• Insulin interacts with insulin receptors which via protein chain
transports GLUT4 to the cell surface to admit glucose into cell
GLUT4
Glycolysis
• Common to virtually all cells –prokaryotic and eukaryotic
• Occurs anaerobically in cell cytoplasm
• Break glycolysis down into 3 steps
• Step 1
• Trap glucose in cell
• Form a 6 membered ring fructose 1,6 biphosphate
• Which can be broken into two 3-C compounds
• Step 2
• Cleave fructose 1,6 biphosphate into 3-C compounds
• Step 3
• Oxidize 3-carbon compounds to pyruvate (NAD+ reduced)
• Generate ATP
• Overall Glucose → 2 pyruvate + 2 ATP
Overview of Glycolysis – Stage 1,2
Step 1 – Trapping Glucose in Cell Via
Phosphoryl Group Transfer
H+ from alcohol
+ H+
Mechanism Phosphorlyation Glucose
• Magnesium ions in
enzyme help make P
more electrophilic
• Enzyme needs a cofactor
– Mg2+
Energy Considerations
The enzyme - kinases
• Kinases transfer phosphoryl groups from a donor
(such as ATP above) to a receptor (such as
glucose)
• In particular if phosphoryl group transferred to a 6
membered ring it is called a hexokinase
• The enzyme undergoes large conformational
changes during reaction (induced fit—see earlier)
The enzyme - kinases
• The cleft holding the glucose is closed
• Need to exclude water so that with hydrophobic R
groups of the enzyme so that PO42- group can
approach
• Don’t want to hydrolyze ATP to ADP and Pi
• Typical KM values for kinases are 0.01 to 0.1 mM
so enzyme is typically at maximum reaction rate
in vivo.
Induced Fit and Water Exclusion
Step 2 -Isomerization Reactions
• Changing between molecules with same formula but different
arrangements of atoms within the molecules
• Glucose to Fructose isomerization practiced in industry to
make sweeteners (Fructose is much sweeter than Glucose)
and it goes into beverages
• Isomerization example:
C5H12
C5H12
C5H12
C5H12
Glucose and Fructose
Glucose – aldehyde sugar
Fructose – ketose sugar
Cα Carbon
Aldehyde-Ketone Isomerization
• We will do this isomerization either forward or backward
twice during the first two phases
• Phase 1: G-6P → F-6P (aldehyde to ketone)
• Phase 2: DHAP → DAP (ketone to aldehyde)
More “electron pushing” -- in Isomerization
a. the -carbon Hydrogen is acidic (electrophilic) due to the
presence of the carbonyl oxygen (and alcohol)
b. carbon can only have four bonds
c. arrows are always drawn from the electron pair to the
atom
d. electrons are pushed to the oxygen since H can only
share 2 electrons in its 1s orbital
e. hydrogen on oxygen are acidic (electrophilic)
f. charges must balance always
g. no atoms can be “lost” since isomers have the same
molecular formula – so H must reattach somewhere
h. Need to regenerate the catalyst
Aldose – Ketose Isomerization
+B :H
2
:B2
B1:
H:B +
H:B11
• Properties of aldehydes due to electron-withdrawing effect
of carbonyl oxygen
• Makes carbonyl carbon susceptible to nucleophilic attack
• Make H on -carbon electophilic
• Even more electrophilic here due to presence of oxygen
substituent on same carbon
Enzyme Catalysts
• Note that B1: and B2: are both bases
• However, when protonated they are acids, B1:H+ and B2:H+
• Analogy is ammonia. When unprotonated it is a weak base;
when protonated it is a weak ammonium acid.
+
Glucose 6 Phosphate → Fructose 6
phosphate
Why the Isomerization?
• Ultimately another phosphate group needs to be added to
•
•
•
•
the molecule so that the 6-membered ring can be cleaved
into two 3-membered rings
It is easier to phosphorylate a primary alcohol than a
secondary alcohol
See table for free energy for this reaction
At cellular conditions, it is slightly exergonic, so Keq not far
from unity
But the following phosphorylation reaction (to make F1,6BP) is heavily exergonic so the reaction will shift to the
right
Alcohols – 1o, 2o 3o
Isomerization to F – 6P
Simplified Isomerization
+H:B
2
B1:
+HB
1
:B2
Phosphoryl Group Transfer
• Phosphofructokinase (PFK) is an allosteric enzyme
• Its activity is very sensitive to the energy status of the cell
• Carbon flux through glycolysis is largely controlled in this
step
• Need two phosphates to insure compound cleaves in
middle to two 3-carbon fragments
• Don’t want a two-carbon and a four carbon – would require
more complex processing
Overall summary
Cleavage
96% in this form
glycerin
Forming DHAP and GAP
How Do 3-Carbon Fragments Fit Into
Glycolysis
• GAP (only 4%) can be can be further oxidized to 1,3 BPG,
whereas DHAP cannot
• Don’t want to waste the DHAP (96%), so nature has
provided an isomerization pathway to convert DHAP into
GAP as shown below:
Forming DHAP and GAP
Acidic H
Isomerization Mechanism-Another view
Stage 3
G-3P Dehydrogenase
Setting Up To Harvest Energy
• So far we’ve invested 2 molecules of ATP –it’s time to get some
back
• Start with the transformation of GAP to 1,3 BPG:
• (note both Pi = HPO42- and OPO32- have 32 e-)
• Top half electrons
12
32
42
2
• NAD+ must be constantly supplied for Glycolysis
to continue. Where do we get it?
• From oxidizing NADH later in metabolism
• How does this reaction work?
0
How’s this done? Two Step Scenario
intermediate
• First reaction (redox) spontaneous
• Second reaction has ∆G > 0 (opposite of phosphoryl hydrolysis)
G-3P to 1,3BPG
His 176
The Intermediate is Stabilized
• Intermediate stabilized by enzyme thioester bond
• Coupling: free energy in oxidation reaction (1st reaction)
drives the second reaction
Why is Thioester bond so energetic?
Harvesting Energy
• For 1,3 BPG → 3-PG + Pi ΔGo’ = -49.3
• For ADP + Pi → ATP
ΔGo’ = +30.5
• Since this is a 3-Carbon molecule, we form 2 moles of ATP
for each 6-carbon glucose fed to Glycolysis
Making Pyruvate
• Reaction 1: rearrangement; Reaction 2: dehydration
Phosphoenolpyruvate
∆Ghydrol = -15.6 kJ/mole
Ghydrol = -61.9 kJ/mole
Keto group more
Stable than enol
3-PG to 2-PG Mechanism
2 PG to PEP
Making Pyruvate
• One PO32- is transferred to ADP (takes energy) get the enol
• Enol is an unstable form of pyruvate which is why PEP has
such a high Ghyd
• Enol converts to more stable keto form
(keto form)
• Glycolysis has now yield a “profit” of two molecules of
ATP/Glucose fed (Remember there are two pyruvates
formed for each glucose fed)
Keto – Enol Tautomerization
H+
Metabolize Pyruvate to Get NAD+
• Use fermentations to oxidize NADH
Yeast cells
Humans
Pyruvate to Ethanol (fermentation)
• Carbon in ethanol is reduced –gains two electrons from NADH
• Oxidation numbers for carbon
• +2
+1
Total valence e-: 18
• Overall: Glucose → Ethanol no net redox
-1
20
Pyruvate to Lactate – Our cells Under
Anaerobic Exercise
• We still must generate NAD+ in order to satisfy the needs
of Glycolysis
•
+2
Total valence e-: 34
0
36
Summary Anaerobic - Lactate
• Used by muscles for
short bursts of energy
if oxygen cannot be
supplied rapidly
enough to body
• Buildup of lactate and
lactic acid causes
fatigue and “burning”
Glycolysis Overall
• Glucose + 2Pi + 2ADP+ 2NAD+ → 2 Pyruvate+ 2ATP
+2NADH +2H+ + 2H2O
• Thus 1 molecule of Glucose has yielded
• Two molecules of pyruvate
• 2 molecules (net) of ATP
• Free energy change: -96 kJ/mole
• Generated 2 molecules ATP at 30.5 kJ/mole and 2 moles of a
reduced species, NADH
• Potential total free energy change possible -2879 kJ/mole
• Process thus far occurred anaerobically
• In other words no O2 molecules were used as oxidants
• For process to continue must oxidize NADH and send it
back as NAD+
Steady-State Concentrations of
Glycolytic Metabolites in Erythrocytes
Metabolite
mM
Glucose
5.0
Glucose-6-phosphate
0.083
Fructose-6-phosphate
0.014
Fructose-1,60.031
bisphosphate
Dihydroxyacetone
0.14
phosphate
Glyceraldehyde-30.019
phosphate
1,30.001
Bisphosphoglycerate
2,34.0
Bisphosphoglycerate
3-Phosphoglycerate
0.12
2-Phosphoglycerate
0.030
Phosphoenolpyruvate
0.023
Pyruvate
0.051
Lactate
2.9
ATP
1.85
ADP
0.14
Pi
1.0
Regulation of Glycolysis
∆Go: offers little insight as to control points
∆G: Steps 2 and 4-9 ∆G ≈ 0 (at equilbrium) control points steps 1,3,10,11
Aerobic Metabolism
• Pyruvate converted into acetyl CoA and enters the Citric
Acid (aka Krebs) cycle.
• The NAD+ required for glycolysis is regenerated from
NADH in the Electron Transport Chain (ETC)
Glycolysis Control – Muscle Cell
• Three enzymes are used to control Glycolysis
• Step 1: Hexokinase (phosphorylation of glucose)
• Step 3: Phosphofructosekinase (mono to di phospho frutose)
• Step 10: Pyruvatekinase (Phosphoenolpyruvate to pyruvate)
• Irreversible and operate far from equilibrium
• Glycolysis is stimulated when energy charge falls
• Energy charge
[ ATP ]  1 / 2[ ADP]
Energy Ch arg e 
[ ATP ]  [ ADP]  [ AMP]
Glycolysis Control – Muscle Cell
• Hexokinase
• Inhibited by product – glucose 6-phosphate (G-6P)
• Glucose 6-phosphate in equilib with Fructose 6 –phosphate (F-6P)
• Phosphofructokinase inhibition will cause F-6P to rise which also
incr G-6P
• However other sugars (such as fructose) bypass this step so it
should not be only control
• The inhibition of F-6P leads to inhibition in G-6P
Glycolysis Control – Muscle Cell
• Phosphofructokinase
• Most important regulator in Glycolysis
• ATP binds to an allosteric (Greek: other object) site on the enzyme
• AMP competes for the site with ATP; AMP has no inhibitory effect
• When ATP/AMP ratio lowered → increases enzyme activity
• Also inhibited by a decrease in pH (such as when lactic acid is
formed; don’t want to damage cell with acid buildup)
Glycolysis Control Muscle
• ATP allosterically hindered
Glycolysis Control – Other Cells
• Liver control steps are similar to those of muscle cell
(steps 1,3,10) but mechanisms are different
• Glucokinase in liver acts as hexokinase in muscle
• Phosphofructokinase responds to signal molecule F-2,6,-BP
• Pyruvatekinase inhibited by ATP at low glucose levels
• But liver has additional functions
• Glucose ↔ glycogen (a polymer of Glucose that can be utilized for
rapid energy)
• Cells have transporter molecules that bring Glucose into
and out of cells (GLUT1-GLUT5)
• Can bring glucose into different cells as a function of their KM value
Glycolysis and Cancer
• Tumor cells rapidly convert glucose to lactic acid even in
the presence of oxygen (called the Warburg effect)
• This glucose uptake is characteristic of aggressive
cancers
• What is the biochemistry reasoning behind this?
• Lactic acid inhibits the immune system from attacking cancer cells
• Cancer cells grow more rapidly than blood vessels supplying them
so that they have adapted to grow under low oxygen conditions

Industrial and Commercial Applications

Food Industry
~ Beer
~ Bread
~ Cheese
~ Wine
~ Yogurt

Pharmaceutical Industry
~ Insulin
~ Vaccine Adjuvants

Energy
~ Fuel Ethanol
Theory
x Biomass
Approx 1st order kinetics
The plot showing the trends for yeast cell growth over time
Lineweaver-Burk Rearrangement

ss 

k
s

s 
 s
 (s)   m  
Michaelis-Menten like kinetics
1
(s)

ks  s ks 1 1

 
m s
m s m
Fermentation Overview
• Yeast species: Saccharomyces cerevisiae converts
carbohydrates (glucose: a monosaccharide sugar) into
CO2 and EtOH
• Follow cell growth with CO2 produced
• More sophisticated ways are measuring yeast cells by
light scattering in a spectrophotometer
• Could also do studies on effect of glucose concentration
on cell growth
Experiment
Resazurin turns pink in the presence of oxygen and colorless in the absence
of oxygen