Glycolysis

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Chapter 18

• Glycolysis
• Biochemistry
• by
• Reginald Garrett and Charles Grisham

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18.1 What Are the Essential Features of
Glycolysis?

• The Embden-Meyerhof (Warburg) Pathway Glycolysis
• Consists of two phases
• First phase converts glucose to two
  Glyceraldehyde-3-P
• Energy investment phase
• Consumes 2 molecules of ATP
• Second phase produces two pyruvates
• Energy generation phase
• Produces 4 molecules of ATP
• Products are 2 pyruvate, 2 ATP and 2 NADH
• Essentially all cells carry out glycolysis
• Ten reactions - same in all cells - but rates
  differ

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Figure 18.1The glycolytic pathway.
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Figure 18.2 Pyruvate produced in glycolysis can
be utilized by cells in several ways. In
animals, pyruvate is normally converted to
acetyl-coenzyme A, which is then oxidized in the
TCA cycle to produce CO2. When oxygen is
limited, pyruvate can be converted to lactate.
Alcoholic fermentation in yeast converts pyruvate
to ethanol and CO2.
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18.2 Why Are Coupled Reactions Important in
Glycolysis?

• Coupled reactions convert some, but not all, of
  the metabolic energy of glucose into ATP
• The free energy change for the conversion of
  glucose to two lactates is -1863.6
• Glucose ? 2 lactate 2H
  DG0 -183.6 kJ/mol
• The production of two molecules of ATP in
  glycolysis is an energy-required process
• 2 ADP 2 Pi ? 2 ATP 2 H2O
  DG0 61 kJ/mol

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• Glycolysis couples these two reactions
• Glucose ? 2 lactate 2H DG0
  -183.6 kJ/mol
• 2 ADP 2 Pi ? 2 ATP 2 H2O
  DG0 61 kJ/mol
• 
  
• Glucose 2 ADP 2 Pi ? 2 lactate 2 ATP 2H
  2 H2O
• DG0 -122.6 kJ/mol
• More than enough free energy is available in the
  conversion of glucose into lactate to drive the
  synthesis of two molecules of ATP

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18.3 What Are the Chemical Principles and
Features of the First Phase of Glycolysis?
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Under cellular condition DG DGo RT ln (
G-6-PADP / GluATP )
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Phase 1

1. Phosphorylation (Hexokinase)
2. Isomerization (Phosphoglucoisomerase)
3. Phosphorylation (Phosphofructokinase)
4. Cleavage (Aldolase)
5. Isomerization (Triose phosphate isomerase)

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Reaction 1 Hexokinase

• Phosphorylation of glucose
• Hexokinase (or glucokinase in liver)
• This is a priming reaction - ATP is consumed here
  in order to get more later
• ATP makes the phosphorylation of glucose
  spontaneous
• Mg2 is required

Mg2
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• The cellular advantages of phosphorylating
  glucose
• Phosphorylation keeps the substrate in the cell
• Keeps the intracellular concentration of glucose
  low, favoring diffusion of glucose into the cell
• Makes it an important site for regulation

Figure 18.4 Glucose-6-P cannot cross the plasma
membrane.
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Hexokinase 1st step in glycolysis ?G large,
negative

• Km for glucose is 0.1 mM cell has 4 mM glucose,
  so hexokinase is normally active
• Hexokinase is regulated --allosterically
  inhibited by (product) glucose-6-P -- but is not
  the most important site of regulation of
  glycolysisWhy? (Figure 18.5)
• Can phosphorylate a variety of hexose sugars,
  including glucose, mannose, and fructose

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Figure 18.5 Glucose-6-phosphate is the branch
point for several metabolic pathways.
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Glucokinase

• The isozymes of hexokinase
• Hexokinase I in brain
• Hexokinase I (75, 0.03mM) and II (25, 0.3mM) in
  muscle
• Glucokinase in liver and pancreas
• Glucokinase (Kmglucose 10 mM) only turns on
  when cell is rich in glucose
• Is not product inhibited
• Is an inducible enzyme by insulin

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Induced fit model (fig 13.24)
Figure 18.7 (a) Mammalian hexokinase I
Figure 18.6 The (a) open and (b) closed states
of yeast hexokinase
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Reaction 2 Phosphoglucoisomerase

• Glucose-6-P (aldose) to Fructose-6-P (ketose)
• Why does this reaction occur
• next step (phosphorylation at C-1) would be tough
  for hemiacetal -OH, but easy for primary -OH
• isomerization activates C-3 for cleavage in
  aldolase reaction
• Phosphoglucose isomerase or glucose phosphate
  isomerase
• Ene-diol intermediate in this reaction

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Phosphoglucoisomerase, with fructose-6-P (blue)
bound.
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Figure 18.8 The phosphoglucoisomerase mechanism
involves opening of the pyranose ring (step 1),
proton abstraction leading to enediol formation
(step 2), and proton addition to the double bond,
followed by ring closure (step 3)
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Reaction 3 Phosphofructokinase

• PFK is the committed step in glycolysis
• The second priming reaction of glycolysis
• Committed step and large, negative DG -- means
  PFK is highly regulated

Fructose-6-P Pi ? Fructose-1,6-bisP DGo
16.3 kJ/mol
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Regulation of Phosphofructokinase

• ATP also is a allosteric inhibitor
• Has two distinct binding sites for ATP (A
  high-affinity substrate site and a low-affinity
  regulatory site)
• AMP reverses the inhibition due to ATP
• Raise dramatically when ATP decrease
• Citrate is also an allosteric inhibitor
• Fructose-2,6-bisphosphate is allosteric activator
  
• PFK increases activity when energy status is low
• PFK decreases activity when energy status is high

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Figure 18.9 At high ATP, phosphofructokinase
(PFK) behaves cooperatively and the activity plot
is sigmoid.
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Figure 18.10 Fructose-2,6-bisphosphate activates
phosphofructokinase, increasing the affinity of
the enzyme for fructose-6-phosphate and restoring
the hyperbolic dependence of enzyme activity on
substrate concentration.
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Figure 18.11 Fructose-2,6-bisphosphate decreases
the inhibition of phosphofructokinase due to ATP.
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Reaction 4 Fructose Bisphosphate Aldolase

• C6 is cleaved to 2 C3s (DHAP, Gly-3-P)
• Fructose bisphosphate aldolase cleaves
  fructose-1,6-bisphosphate between the C-3 and C-4
  carbons to yield two triose phosphates
• Dihydroxyacetone phosphate (DHAP) and
  glyceraldehyde-3-phosphate (G-3-P)

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The aldolase reaction is unfavorable as written
at standard state. The cellular ?G, however, is
close to zero.
The aldolase reaction in glycolysis is merely the
reverse of the aldol condensation well known to
organic chemists.
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• Animal aldolases are Class I aldolases
• Class I aldolases form covalent Schiff base
  intermediate between substrate and active site
  lysine

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• Class II aldolase are produced mainly in bacteria
  and fungi

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Reaction 5 Triose Phosphate Isomerase

• Only G-3-P goes directly into the second phase,
  DHAP must be converted to G-3-P
• Triose phosphate isomerase
• An ene-diol mechanism
• Active site Glu acts as general base
• is a near-perfect enzyme (Table 13.5)

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Figure 18.13 A reaction mechanism for triose
phosphate isomerase. In the yeast enzyme, the
catalytic residue is Glu165.
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18.4 What Are the Chemical Principles and
Features of the Second Phase of Glycolysis?

• Metabolic energy produces 4 ATP
• Net ATP yield for glycolysis is two ATP
• Second phase involves two very high energy
  phosphate intermediates
• 1,3 BPG
• Phosphoenolpyruvate
• Substrate-level phosphorylation

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Phase 2

1. Oxidation and Phosphorylation (Glyceraldehyde-3-P
   Dehydrogenase)
2. Substrate-level Phosphorylation (phosphoglycerate
   kinase)
3. Isomerization (Phosphoglycerate isomerase)
4. Dehydration (Enolase)
5. Substrate-level Phosphorylation (pyruvate kinase)

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Reaction 6 Glyceraldehyde-3-Phosphate
Dehydrogenase

• G-3-P is oxidized to 1,3-BPG
• Energy yield from converting an aldehyde to a
  carboxylic acid is used to make 1,3-BPG and NADH
• Oxidation (aldehyde to carboxylic acid) and
  phosphorylation

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G3PDH (or GAPDH)

• Mechanism involves covalent catalysis and a
  nicotinamide coenzyme

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Reaction 7 Phosphoglycerate Kinase

• ATP synthesis from a high-energy phosphate
• This is referred to as "substrate-level
  phosphorylation"
• Coupled reactions 6th and 7th reactions
• Glyceraldehyde-3-P ADP Pi NAD ?
• 3-phosphoglycerate ATP NADH H
  DGo -12.6 kJ/mol

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• ATP is synthesized by three major routes
• Substrate-level phosphorylation (Glycolysis,
  Citric acid cycle)
• Oxidative phosphorylation (Driven by electron
  transport)
• Photophosphorylation (Photosynthesis)

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• 2,3-BPG (for hemoglobin) is made by circumventing
  the PGK reaction
• Bisphosphoglycerate mutase
• Erythrocytes contain 4-5 mM 2,3-BPG

Figure 18.16 The mutase that forms 2,3-BPG from
1,3-BPG requires 3-phosphoglycerate.
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Reaction 8 Phosphoglycerate Mutase

• Repositions the phosphate
• Mutase catalyzes migration of a functional group
  within a substrate

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• Phosphoenzyme intermediates
• A bit of 2,3-BPG is required as a cofactor

The catalytic His183 at the active site of E.
coli phosphoglycerate mutase
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Reaction 9 Enolase

• The formation of PEP from 2-PG
• Dehydration
• Make a high-energy phosphate in preparation for
  ATP synthesis in step 10 of glycolysis

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Reaction 10 Pyruvate Kinase

• The pyruvate kinase reaction converts PEP to
  pyruvate, driving synthesis of ATP.
• Substrate-level phosphorylation
• Another key control point for glycolysis
• Enol-keto tautomer

.
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Figure 18.19 The conversion of
phosphoenolpyruvate (PEP) to pyruvate may be
viewed as involving two steps phosphoryl
transfer, followed by an enol-keto
tautomerization. The tautomerization is
spontaneous and accounts for much of the free
energy change for PEP hydrolysis.
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• Large, negative ?G -- regulation
• Allosteric regulation
• Activated by AMP, F-1,6-bisP
• Inhibited by ATP ,acetyl-CoA, and alanine
• Liver pyruvate kinase is regulated by covalent
  modification
• Responsive to hormonally-regulated
  phosphorylation in the liver (glucagon)
• The phosphorylated form of the enzyme is more
  strongly inhibited by ATP and alanine.
• Has a higher Km for PEP

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18.5 What Are the Metabolic Fates of NADH and
Pyruvate Produced in Glycolysis?

• Aerobic or anaerobic?
• NADH must be recycled to NAD
• If O2 is available, NADH is re-oxidized in the
  electron transport pathway, making ATP in
  oxidative phosphorylation (chapter 20)
• In anaerobic conditions, NADH is re-oxidized by
  lactate dehydrogenase (LDH), providing additional
  NAD for more glycolysis

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Figure 18.21 (a) Pyruvate reduction to ethanol in
yeast provides a means for regenerating NAD
consumed in the glyceraldehyde-3-P dehydrogenase
reaction. (b) In oxygen-depleted muscle, NAD is
regenerated in the lactate dehydrogenase reaction.
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• Pyruvate also has two possible fates
• aerobic into citric acid cycle (chapter 19)
  where it is oxidized to CO2 with the production
  of additional NADH (and FADH2)
• anaerobic (fermentation)
• In yeast reduced to ethanol
• Pyruvate decarboxylase (TPP)
• Alcohol dehydrogenase (Reoxidized NADH to NAD)
• In animals reduced to lactate
• Lactate dehydrogenase (Reoxidized NADH to NAD)

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18.6 How Do Cells Regulate Glycolysis?

• The elegant evidence of regulation (See Figure
  18.22)
• Standard state ?G values are variously positive
  and negative
• ?G in cells is revealing
• Most values near zero (reactions 2 and 4-9)
• 3 of 10 Reactions have large, negative ?G
• Large negative ?G Reactions are sites of
  regulation
• Hexokinase
• Phosphofructokinase
• Pyruvate kinase

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Overview of the regulation of glycolysis.
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18.7 Are Substrates Other Than Glucose Used in
Glycolysis?

• Sugars other than glucose can be glycolytic
  substrates
• Fructose and mannose are routed into glycolysis
  by fairly conventional means.

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• Fructose
• In liver
• Fructokinase
• Fructose ATP ? fructose-1-phosphate ADP H
• Fructose-1-phosphate aldolase
• fructose-1-phosphate ? glyceraldehyde DHAP
• Triose kinase
• glyceraldehyde ? glyceraldehyde-3-phosphate
• In kidney and muscle
• Hexokinase
• Fructose ATP ? fructose-6-phosphate ADP H

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• Mannose
• Hexokinase
• mannose ATP ? mannose-6-phosphate ADP H
• Phosphomannoisomerase
• mannose-6-phosphate ? fructose-6-phosphate
• Galactose is more interesting - the Leloir
  pathway "converts" galactose to glucose
• Galactokinase
• Galactose ATP ? galactose-1-phosphate ADP
  H
• Galactose-1-phosphate uridylyltransferase
• Phosphoglucomutase
• UDP-galactose-4-epimerase

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Figure 18.24 Galactose metabolism via the Leloir
pathway.
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Figure 18.25 The galactose-1-phosphate
uridylyltransferase reaction involves a
ping-pong kinetic mechanism.
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• Galactosemia
• Defects in galactose-1-P uridylyltransferase
• Galactose accumulate causes cataracts and
  permanent neurological disorders
• In adults, UDP-glucose pyrophosphorylase also
  works with galactose-1-P, reducing galactose
  toxicity

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• Lactose Intolerance
• The absence of the enzyme lactase
  (b-galactosidase)
• Diarrhea and discomfort

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• Glycerol can also enter glycolysis
• Glycerol is produced by the decomposition of
  triacylglycerols (chapter 23)
• Converted to glycerol-3-phosphate by the action
  of glycerol kinase
• Then oxidized to DHAP by the action of glycerol
  phosphate dehydrogenase
• NAD as the required coenzyme

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18.8 How Do Cells Respond to Hypoxic Stress?

• Glycolysis is an anaerobic pathwayit does not
  require oxygen
• The TCA (tricarboxylic acid) cycle is aerobic.
  When oxygen is abundant, cells prefer to combine
  these pathways in aerobic metabolism
• When oxygen is limiting, cells adapt to carry out
  more glycolysis

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• Hypoxia (oxygen limitation) causes changes in
  gene expression that increases
• Angiogenesis (the growth of new blood vessels)
• Synthesis of red blood cells
• Levels of some glycolytic enzymes (a high rate of
  glycolysis)
• Hypoxic stress
• A trigger for this is a DNA binding protein
  called hypoxia inducible factor (HIF)
• HIF is regulated at high oxygen levels by
  hydroxylase factor-inhibiting HIF (FIH-1)

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• Hypoxia inducible factor (HIF)
• A heterodimer consists of two subunits
• A constitutive, stable nuclear b subunit HIF-1ß
• An inducible, unstable hypoxia-responsive HIF-a
  subunit
• Bind to the hypoxia responsive element (HRE) of
  hypoxia-inducible genesActivating transcription
  of these genes
• HIF-a regulation is a multistep process
• Gene splicing
• Acetylation (Inhibited)
• Hydroxylation (Inhibited)
• Phosphorylation (activated)

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• Under normal oxygen levels, HIF-a are synthesized
  but quickly degraded
• When oxygen is plentiful, HIF-1a is hydroxylated
  by the prolyl hydroxylases (PHDs)
• These hydroxylation ensure its binding to
  ubiquitin E3 ligase, which leads to rapid
  proteolysis
• HIF-1a binding to the ubiquitin E3 ligase is also
  promoted by acetylation by the ARD1
  acetyltransferase
• FIH-1 (hydroxylase factor-inhibiting HIF-a)
  hydroxylates HIF-a at Asn803
• PHDs and FIH-1 both are oxygen-dependent

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Figure 18.28