Download Glycolysis 1

Glycolysis 1
Lecture 25
Key Concepts
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Overview of the Glycolytic Pathway
– Glycolysis generates a small amount of ATP
– Preview of the ten enzyme-catalyzed reactions of glycolysis
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Stage 1: ATP Investment
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Reaction 1: Hexokinase
Reaction 2: Phosphoglucose isomerase
Reaction 3: Phosphofructokinase
Reaction 4: Aldolase
Stage 2: Formation of GAP
– Reaction 5: Triose phosphate isomerase
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Stage 3: ATP Earnings
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Reaction 6: Glyceraldehyde-3-P dehydrogenase
Reaction 7: Phosphoglycerate kinase (substrate level phosphorylation)
Reaction 8: Phosphoglycerate mutase
Reaction 9: Enolase
Reaction 10: Pyruvate kinase (substrate level phosphorylation)
Overview of the Glycolytic Pathway
Glycolysis is considered one of the core metabolic pathways in nature for
three primary reasons
1. glycolytic enzymes are highly conserved among all living organisms,
suggesting it is an ancient pathway.
2. glycolysis is the primary pathway for ATP generation under anaerobic
conditions and in cells lacking mitochondria such as erythrocytes.
3. metabolites of glycolysis are precursors for a large number of
interdependent pathways, including mitochondrial ATP synthesis.
Pathway 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
∆Gº’ = -35.8 kJ/mol
Pathway Questions
3. What are the key regulated enzymes in glycolysis?
Hexokinase, Phosphofructokinase, Pyruvate kinase
4. What are examples of glycolysis in real life?
A deficiency in the hexokinase-related enzyme, glucokinase,
leads to a rare form of diabetes, which is caused by the inability
of liver and pancreatic cells to phosphorylate glucose inside cells
when blood glucose levels are elevated.
The complete oxidation of glucose to CO2 and H2O
is summarized by the reaction:
Glucose (C6H12O6) + 6O2 → 6CO2 + 6H2O
∆Gº’ = -2,840 kJ/mol
∆G = -2,937 kJ/mol
Glucose → CO2 + H2O
Fermentation
Lactobacillus
Convert glucose
into lactic acid
and CH4 and H2
Glycolysis has 3 Stages
ATP Investment
GAP formation and TIM
ATP Earnings
2 ATP used
Fructose-1,6-bisphosphate
Net result: 2 molecules of GAP
Each molecule of GAP
4 ATP gained
Net: 2 ATP
Glycolysis produces a
small amount of ATP
More importantly:
glycolysis produces
Pyruvate
What is the most important product of
glycolysis?
What is the most important intermediate
metabolite 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
Important features of glycolysis
• Ten enzymatic reactions
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primarily bond rearrangements
phosphoryl transfer reactions
isomerizations
an aldol cleavage
an oxidation
a dehydration
• There is no net loss of carbon or oxygen atoms.
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.
Free energy changes for the ten glycolytic reactions
STAGE 1: ATP INVESTMENT
Stage 1 of glycolysis includes three enzymatic
reactions that accomplish two tasks:
1) Use ATP as the phosphate donor to create
phosphorylated compounds that are negatively
charged and cannot diffuse out of the cell
2) Prepare the backbone of glucose to be cleaved
Reaction 1: Phosphorylation of glucose by
hexokinase or glucokinase
hexokinase is found
in all cells
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 C6 carbon of glucose
Hexokinase is feedback inhibited by glucose-6-P which binds
to a regulatory site in the amino terminus of the enzyme
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 multi-step pathway that
involves opening and closing of the ring structure
Reaction 3: Phosphorylation of fructose-6-P
to fructose-1,6-BP by phosphofructokinase
Reaction 3 is the second ATP investment reaction in glycolysis and involves
the coupling of ATP hydrolysis to a phosphoryl transfer reaction that is
catalyzed by the enzyme phosphofructokinase
STAGE 2: BACKBONE CLEAVAGE AND
GAP FORMATION
Stage 2 of glycolysis includes two enzymatic
reactions that produce two molecules of
glyceraldehyde-3-phosphate from every
molecule of glucose that enters glycolysis
Reaction 4: Cleavage of fructose-1,6-BP into
glyceraldehyde-3-P and dihydroxyacetone-P
by aldolase
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
Glyceraldehyde-3-P, not dihydroxyacetone-P, is the substrate for reaction 6 in
the glycolytic pathway.
The enzyme triose phosphate isomerase converts the ketose
dihydroxyacetone-P to the aldose glyceraldehyde-3-P in an isomerization
reaction.
The TIM barrel structure was named for this
enzyme (triose phosphate isomerase)
Why is this protein considered to
be a kinetically perfect enzyme?
STAGE 3: ATP EARNINGS
Three key features of stage 3 reactions need to be pointed out :
1. two substrate level phosphorylation reactions catalyzed by the
enzymes phosphoglycerate kinase and pyruvate kinase generate a
total of 4ATPs (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 to form
1,3-bisphosphoglycerate by glyceraldehyde3-P dehydrogenase
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.
glyceraldehyde-3-P dehydrogenase
• Requires NAD+
• NAD+ must be continually replenished within the
cytosol
– aerobically in the mitochondrial matrix by the electron
transport chain
– anaerobically in the cytosol by the enzyme lactate
dehydrogenase which converts pyruvate to lactate
– anaerobically in yeast using the enzyme alcohol
dehydrogenase which converts pyruvate to CO2 and
ethanol
1,3-bisphosphoglycerate has a standard free energy of
hydrolysis that is higher than ATP hydrolysis
This difference in free energies is harnessed by the enzyme
phosphoglycerate kinase in reaction 7 to drive the synthesis of
ATP by a mechanism called substrate level phosphorylation.
Reaction 7: Substrate level phosphorylation
to generate ATP in the conversion of
1,3-bisphosphoglycerate to 3phosphoglycerate by phosphoglycerate
kinase
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.
The molecular structure of
phosphoglycerate kinase is
similar to hexokinase in that it
has two lobes (jaws) that each
bind one of the substrates
(ADP-Mg2+ or 1,3bisphosphoglycerate) leading
to a large conformational
change in the enzyme that
brings the substrates close
together and excludes H2O
from the active site.
Reactions 6 and 7 are coupled reactions!
(Rxn 6)
Glyceraldehyde-3-P + Pi + NAD+ →
1,3-bisphosphoglycerate + NADH + H+
∆Gº’ = +6.3 kJ/mol ∆G = -1.3 kJ/mol
(Rxn 7)
1,3-bisphosphoglycerate + ADP →
3-phosphoglycerate + ATP
∆Gº’ = -18.9 kJ/mol ∆G = +0.1 kJ/mol
(Coupled)
Glyceraldehyde-3-P + Pi + ADP + NAD+ →
3-phosphoglycerate + ATP + NADH + H+
∆Gº’ = -12.6 kJ/mol ∆G = -1.2 kJ/mol
Actual change in free energy for each of these two reactions is very close to
zero, and therefore both reactions are in fact reversible inside the cell. This is
important for gluconeogenesis.
Reaction 8: Phosphoryl shift in 3phosphyglycerate to form 2phosphoglycerate by phosphoglycerate
mutase
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 that generates ATP earnings in
step 10.
mechanism of this highly reversible reaction requires a
phosphoryl transfer from a phosphorylated histidine residue
(His-P) located in the enzyme active site
BPG can diffuse out of
active site.
Remember that it (BPG)
is important in regulation
of Hemoglobin binding
of oxygen
Reaction 9: Dehydration of 2phosphoglycerate to form
phosphoenolpyruvate by enolase
Standard free energy for this reaction is relatively small (∆Gº’ = +1.7 kJ/mol),
meaning that the overall metabolic energy available from 2-phosphoglycerate
and phosphoenolpyruvate is similar.
But, 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 ∆Gº’ = -16 kJ/mol, whereas for phosphoenolpyruvate it is an incredible ∆Gº’
= -62 kJ/mol !
Reaction 10: Substrate level
phosphorylation to generate ATP in the
conversion of phosphoenolpyruvate to
pyruvate by pyruvate kinase
The second of two substrate level phosphorylation reactions in glycolysis
that couples energy released from phosphate hydrolysis (∆Gº’ = -62 kJ/mol) to
that of ATP synthesis (∆Gº’ = +30.5 kJ/mol). Unlike phosphoenolpyruvate,
pyruvate is a stable compound in cells that is utilized by many other metabolic
pathways as will be described later.