Glycolysis 1:
Glycolysis consists of two stages, an ATP
investment stage, and an ATP earnings stage
Bioc 460 Spring 2008 - Lecture 25 (Miesfeld)
Lactate build-up
can limit exercise
Metabolism of glucose by yeast under anaerobic
conditions leads to the production of ethanol and CO2
Key Concepts in Glycolysis
•
Metabolic pathways consist of a series of enzymatic reactions that share
reactants and products. Metabolic flux (rate of metabolite interconversion) is
highly-regulated by both enzyme activity and substrate availability. Nutrient
availability affects metabolic flux.
•
Glycolysis is an ancient pathway that cleaves glucose (C6H12O6) into two
molecules of pyruvate (C3H3O3). Under aerobic conditions, the pyruvate is
completely oxidized by the citrate cycle to generate CO2, whereas, under
anaerobic (lacking O2) conditions, it is either converted to lactate, or to ethanol +
CO2 (fermentation).
•
The glycolytic pathway consists of ten enzymatic steps organized into two
stages. In Stage 1, two ATP are invested to “prime the pump,” and in Stage 2,
four ATP are produced to give a net ATP yield of 2 ATP/glucose.
•
Three glycolytic enzymes catalyze highly exergonic reactions (G<<0) which
drive metabolic flux through the pathway; these enzymes are regulated by the
energy charge in the cell (ATP requirements). The three enzymes are
hexokinase, phosphofructokinase 1, and pyruvate kinase.
Glucose metabolism in the liver before breakfast
Glucagon signaling in liver cells
activates both a catabolic
pathway (glycogen degradation)
and an anabolic pathway
(gluconeogenesis), while at the
same time inhibiting the
catabolism of glucose by the
glycolytic pathway.
Glucose metabolism in the liver after breakfast
Within an hour of eating a bowl of
cereal and drinking a cup of fruit
juice, your insulin levels increase
due to elevated blood glucose
causing activation of the insulin
signaling pathway and
stimulation of glucose uptake,
glycogen synthesis, and an
increase in glucose catabolism
by the glycolytic pathway.
Metabolic pathways are linked sets of enzymatic
reactions that share common intermediates
Remember that substrate
concentration and enzyme
activity levels affect flux
through metabolic pathways
There are six major groups of metabolic
pathways in nature
1. Anaerobic and aerobic respiration
2. Photosynthesis and carbon fixation
3. Carbohydrate metabolism
4. Lipid metabolism
5. Amino acid metabolism
6. Nucleotide metabolism
•
Metabolic pathways are highly interdependent and exquisitely controlled by
substrate availability and enzyme activity levels.
•
Key to understanding metabolic integration in terms of nutrition, exercise,
and disease (e.g., diabetes and obesity) is learning how metabolic flux
between pathways is regulated and controlled.
http://www.expasy.org/cgi-bin/show_thumbnails.pl
Should you memorize this chart?
Hierarchical Nature of
Metabolism
Four classes of
macromolecules (proteins,
nucleic acids, carbohydrates,
and lipids)
Six primary metabolite
groups (amino acids,
nucleotides, fatty acids,
glucose, pyruvate, acetyl
CoA)
Seven small biomolecules
(NH4+, CO2, NADH, FADH2,
O2, ATP, H2O)
Note that you can download a PDF file
of this metabolic map from the study
guides page on the website.
The six major groups of
pathways can be
subdivided into those
responsible for energy
conversion, and those
involved in the
synthesis and
degradation of
macromolecules.
Moreover, the major
groups themselves
consist of several
metabolic modules, all
of which will be
examined individually.
We will use the “divide
and conquer” strategy to
study metabolism, starting
with energy conversion
pathways, and then
examining the synthesis
and degradation of
carbohydrates, lipids, and
amino acids. Nucleotide
metabolism is taught in
other courses.
The last two lectures in
this course (lectures 40
and 41) cover metabolic
regulation using diet,
exercise, and diabetes as
conceptual themes.
Four central questions to keep in mind when you
study each metabolic pathway
1. What does the pathway accomplish for the cell?
2. What is the overall net reaction of the pathway?
3. What are the key regulated enzymes in the pathway?
4. What are examples of this pathway in real life?
Glycolysis: Let’s Answer the Four Questions
1. What does glycolysis accomplish for the cell?
– Generates a small amount of ATP which is critical under
anaerobic conditions.
– Generates pyruvate, a precursor to acetyl CoA, lactate, and
ethanol (in yeast).
2. What is the overall net reaction of glycolysis?
Glucose + 2NAD+ + 2ADP + 2 Pi →
2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O
ΔGº’ = -35.5 kJ/mol
The Four Metabolic Pathway Questions
3. What are the key regulated enzymes in glycolysis?
Hexokinase, Phosphofructokinase 1, Pyruvate kinase
4. What are examples of glycolysis in real life?
Glycolysis is the sole source of ATP under anaerobic conditions
which can occur in animal muscle tissue during intense exercise.
Fermentation also relies on glycolysis which is a process that is
used to make alcoholic beverages when yeast cells are provided
glucose without oxygen.
Where does glycolysis fit into the metabolic map?
Glycolysis is a central
pathway that takes
glucose generated by
carbohydrate metabolism
and converts it to
pyruvate. Under aerobic
conditions, the pyruvate is
oxidized in the citrate
cycle which generates
reducing power for redox
reactions in the electron
transport system that
result in ATP production
by oxidative
phosphorylation.
Glycolysis takes
place entirely in
the cytosol,
whereas, pyruvate
oxidation occurs in
the mitochondrial
matrix where ATP
is generated.
Oxygen is not
required for
glycolysis in the
cytosol (anaerobic)
but it is necessary
for aerobic
respiration in the
mitochondrial
matrix where the
O2 serves as the
terminal electron
acceptor.
The complete oxidation of glucose to CO2 and
H2O is highly favorable and releases a large amount
of energy that can be harnessed for ATP synthesis
Glucose (C6H12O6) + 6O2 → 6CO2 + 6H2O
ΔGº’ = -2,840 kJ/mol
ΔG = -2,937 kJ/mol
ΔGº’ for ATP synthesis = -30.5 kJ/mol
ΔG for ATP synthesis = ~-50 kJ/mol
Theoretical maximum yield = ~60 ATP/glucose
Actual yield = 32 ATP/glucose
Why are only 32 ATP generated out of a possible ~60 ATP?
Pyruvate can also be converted anaerobically to
ethanol and CO2 by fermentation in some
micoroorganisms, or converted to lactate
Overview of the Glycolytic Pathway
For every mole of glucose
entering glycolysis, two moles of
glyceraldehyde-3-P (GAP) are
metabolized to pyruvate,
generating in the process a net 2
ATP and 2 NADH.
The NADH is a source of
reducing power for the cell.
The two stages of glycolysis
The ATP investment stage
generates the high energy
intermediate glyceraldehyde-3-P
(GAP) which is then oxidized to
produce NADH and 1,3bisphosphoglycerate. The next
four reactions lead to the
production of FOUR total ATP
because each glucose molecule
results in the production of TWO
pyruvate. The net yield of ATP in
glycolysis is therefore TWO ATP.
Stage 1
• Investment of 2 ATP
• Production of 2
Glyceraldehyde-3-P
(GAP)
• The two highly
regulated steps are
hexokinase and
phosphofructokinase 1
(both respond directly
or indirectly to energy
charge).
Stage 2
• Reducing power is
captured in the form
of NADH; this is a
critical step.
• Phosphoglycerate
kinase and pyruvate
kinase catalyze a
substrate level
phosphorylation
reaction yielding 4
ATP (2 net ATP).
• The two pyruvate
molecules are
further metabolized.
Each molecule of GAP
No loss of carbons
or oxygen in
glycolysis
The six carbons and
six oxygens present
in glucose are
stoichiometrically
conserved by
glycolysis in the two
molecules of
pyruvate that are
produced. Hydrogen
atoms in glucose
are lost as H2O
molecules and in the
reduction of NAD+.
Chemical features of the glycolytic reactions
• Ten enzymatic reactions
– primarily bond rearrangements
– phosphoryl transfer reactions
– isomerizations
– an aldol cleavage
– an oxidation
– a dehydration
• Ideally, you should know the names of all ten enzymes and the
reactants and products. The names describe the metabolite
structures, draw them if you like, or visualize them in your head.
• At the very minimum, you need to know which steps the ATP
hydrolysis and synthesis takes place, the net reaction of glycolysis,
and the three key enzymes the control glycolytic flux.
Free energy changes for the ten glycolytic reactions
Gº’ = -35.5 kJ/mol
G = -72.4 kJ/mol
Reaction 1: Phosphorylation of glucose by
hexokinase or glucokinase
Hexokinase is
found in all cells.
A related enzyme
with same
enzymatic activity,
glucokinase, is
present primarily in
liver and
pancreatic cells.
Hexokinase binds glucose through an induced fit
mechanism that excludes H2O from the enzyme active site
and brings the phosphoryl group of ATP into close proximity
with the C-6 carbon of glucose
Hexokinase is
feedback inhibited by
glucose-6-P which
binds to a regulatory
site in the amino
terminus of the enzyme
Why does it make
sense that
hexokinase is
feedback inhibited by
glucose-6-P when
energy charge in the
cell is high?
Reaction 2: Isomerization of glucose-6-P to
fructose-6-P by phosphoglucose isomerase
Phosphoglucose isomerase (phosphohexose isomerase) interconverts an aldose
(glucose-6-P) and a ketose (fructose-6-P) through a complex reaction mechanism
that involves opening and closing of the ring structure.
Reaction 3: Phosphorylation of fructose-6-P to
fructose-1,6-BP by phosphofructokinase 1
Reaction 3 is the second ATP investment reaction in glycolysis and involves the
coupling of an ATP phosphoryl transfer reaction catalyzed by the enzyme
phosphofructokinase 1 (PFK-1). This is a key regulated step in the glycolytic
pathway because the activity of PFK-1 is controlled by numerous allosteric
effectors (positive and negative).
Reaction 4: Cleavage of fructose-1,6-BP
by aldolase to generate glyceraldehyde-3-P
and dihydroxyacetone-P
The splitting of fructose-1,6-BP into the triose phosphates glyceraldehyde-3-P
and dihydroxyacetone-P is the reaction that puts the lysis in glycolysis (lysis
means splitting).
Reaction 5: Isomerization of dihydroxyacetone-P to
glyceraldehyde-3-P by triose phosphate isomerase
The original TIM
barrel structure
Glyceraldehyde-3-P, rather than dihydroxyacetone-P, is the substrate for
reaction 6 in the glycolytic pathway, making this isomerization necessary.
STAGE 2: ATP EARNINGS
Three key features of the very important stage 2 reactions:
1. Two substrate level phosphorylation reactions catalyzed by the
enzymes phosphoglycerate kinase and pyruvate kinase generate a
total of 4 ATP/glucose (net yield of 2ATP) in stage 2 of glycolysis.
2. An oxidation reaction catalyzed by glyceraldehyde-3-P
dehydrogenase generates 2 NADH molecules that can be shuttled
into the mitochondria to produce more ATP by oxidative
phosphorylation.
3. Reaction 10 is an irreversible reaction that must be bypassed in
gluconeogenesis by two separate enzymatic reactions catalyzed by
pyruvate carboxylase and phosphoenolpyruvate carboxykinase
Reaction 6: Oxidation and phosphorylation of
glyceraldehyde-3-P by glyceraldehyde-3-P
dehydrogenase to form 1,3-bisphosphoglycerate
The glyceraldehyde-3-P dehydrogenase reaction is a critical step in glycolysis
because it uses the energy released from oxidation of glyceradehyde-3-P to
drive a phosphoryl group transfer reaction using inorganic phosphate (Pi) to
produce 1,3-bisphosphoglycerate.
Reaction 7: Generation of ATP by
phosphoglycerate kinase in the conversion of
1,3-bisphosphoglycerate to 3-phosphoglycerate
Phosphoglycerate kinase catalyzes the payback reaction in glycolysis because
it replaces the 2 ATP that were used in stage 1 to prime the glycolytic pathway.
Remember, this occurs twice for every glucose that entered glycolysis. This is
an example of a substrate level [ADP] phosphorylation reaction, i.e., ATP
synthesis that is not the result of aerobic respiration or photophosphorylation.
Reaction 8: Phosphoryl shift by phosphoglycerate
mutase to convert 3-phosphyglycerate
to 2-phosphoglycerate
The purpose of reaction 8 is to generate a compound, 2-phosphoglycerate, that
can be converted to phosphoenolpyruvate in the next reaction, in preparation
for a second substrate level phosphorylation to generate ATP.
The mechanism of this highly reversible reaction requires
a phosphoryl transfer from a phosphorylated histidine
residue (His-P) located in the enzyme active site
The metabolic
intermediate 2,3BPG can diffuse
out of active site
before it is
converted to 2phosphoglycerate.
Remember that
2,3-BPG is
important in the
regulation of
oxygen binding by
hemoglobin.
Reaction 9: Dehydration of 2-phosphoglycerate by
enolase to form phosphoenolpyruvate (PEP)
The standard free energy for this reaction is relatively small (ΔGº’ = +1.7
kJ/mol) but it traps the phosphate group in an unstable enol form, resulting in a
dramatic increase in the phosphoryl transfer potential of the triose sugar.
Standard free energy change for phosphate hydrolysis in 2-phosphoglycerate is
ΔGº’ = -16 kJ/mol, whereas the standard freen energy change for phosphate
hydrolysis of phosphoenolpyruvate it is an incredible ΔGº’ = -62 kJ/mol !
Reaction 10: Generation of ATP by pyruvate kinase
when phosphoenolpyruvate is converted to pyruvate
The second of two substrate level phosphorylation reactions in glycolysis that
couples energy released from phosphate hydrolysis (ΔGº’ = -62 kJ/mol) to that
of ATP synthesis (ΔGº’ = +30.5 kJ/mol). Unlike phosphoenolpyruvate, pyruvate
is a stable compound in cells that is utilized by many other metabolic pathways.
Fill in the names of all glycolytic enzymes and
metabolites based only on the chemical structures