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Transcript Hexokinase

Chapter 18
Reginald Garrett and Charles Grisham
18.1 – What Are the Essential Features
of Glycolysis?
The Embden-Meyerhof (Warburg) Pathway: Glycolysis
• Consists of two phases:
1. First phase converts glucose to two Glyceraldehyde-3-P
– Energy investment phase
– Consumes 2 molecules of ATP
2. 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
Figure 18.1
The glycolytic pathway.
Figure 18.2 Pyruvate produced in glycolysis can be utilized by cells in
several ways. In animals, pyruvate is normally converted to acetylcoenzyme 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.
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
• Glycolysis couples these two reactions
Glucose  2 lactate + 2H+
2 ADP + 2 Pi  2 ATP + 2 H2O
DG0’ = -183.6 kJ/mol
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
18.3 – What Are the Chemical Principles
and Features of the First Phase of
Under cellular condition
DG = DGo’ + RT ln ( [G-6-P][ADP] / [Glu][ATP] )
Phase 1
1. Phosphorylation (Hexokinase)
2. Isomerization (Phosphoglucoisomerase)
3. Phosphorylation (Phosphofructokinase)
4. Cleavage (Aldolase)
5. Isomerization (Triose phosphate isomerase)
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
• Mg2+ is required
The cellular advantages of phosphorylating
1. Phosphorylation keeps the substrate in the cell
2. Keeps the intracellular concentration of glucose
low, favoring diffusion of glucose into the cell
3. Makes it an important site for regulation
Figure 18.4 Glucose6-P cannot cross the
plasma membrane.
1st step in glycolysis; DG 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
glycolysis—Why? (Figure 18.5)
• Can phosphorylate a variety of hexose sugars,
including glucose, mannose, and fructose
Figure 18.5 Glucose-6-phosphate is the branch point for several metabolic pathways.
• 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
Induced fit model
(fig 13.24)
Figure 18.6 The (a) open and (b)
closed states of yeast hexokinase
Figure 18.7 (a) Mammalian
hexokinase I
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
with fructose-6-P (blue)
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)
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
Regulation of Phosphofructokinase
1. ATP also is a allosteric inhibitor
– Has two distinct binding sites for ATP (A highaffinity substrate site and a low-affinity regulatory
2. AMP reverses the inhibition due to ATP
– Raise dramatically when ATP decrease
3. Citrate is also an allosteric inhibitor
4. Fructose-2,6-bisphosphate is allosteric activator
• PFK increases activity when energy status is
• PFK decreases activity when energy status is
Figure 18.9 At high ATP, phosphofructokinase
(PFK) behaves cooperatively and the activity plot
is sigmoid.
Figure 18.10 Fructose2,6-bisphosphate
increasing the affinity
of the enzyme for
and restoring the
hyperbolic dependence
of enzyme activity on
substrate concentration.
Figure 18.11 Fructose2,6-bisphosphate
decreases the inhibition
of phosphofructokinase
due to ATP.
Reaction 4: Fructose Bisphosphate
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
• Dihydroxyacetone phosphate (DHAP) and
glyceraldehyde-3-phosphate (G-3-P)
The aldolase reaction is unfavorable as written at standard state.
The cellular ΔG, however, is close to zero.
The aldolase reaction in
glycolysis is merely the
reverse of the aldol
condensation well known
to organic chemists.
• Animal aldolases are Class I aldolases
– Class I aldolases form covalent Schiff base
intermediate between substrate and active site
• Class II aldolase are produced mainly in
bacteria and fungi
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)
Figure 18.13 A reaction mechanism for triose phosphate isomerase.
In the yeast enzyme, the catalytic residue is Glu165.
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
Phase 2
6. Oxidation and Phosphorylation
(Glyceraldehyde-3-P Dehydrogenase)
7. Substrate-level Phosphorylation
(phosphoglycerate kinase)
8. Isomerization (Phosphoglycerate isomerase)
9. Dehydration (Enolase)
10.Substrate-level Phosphorylation (pyruvate
Reaction 6: Glyceraldehyde-3Phosphate 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
• Oxidation (aldehyde to carboxylic acid) and
• Mechanism involves covalent catalysis and a
nicotinamide coenzyme
Reaction 7: Phosphoglycerate Kinase
ATP synthesis from a high-energy phosphate
• This is referred to as "substrate-level
• Coupled reactions; 6th and 7th reactions
Glyceraldehyde-3-P + ADP + Pi + NAD+ →
3-phosphoglycerate + ATP + NADH + H+ DGo’= -12.6 kJ/mol
ATP is synthesized by three major routes:
1. Substrate-level phosphorylation
(Glycolysis, Citric acid cycle)
2. Oxidative phosphorylation (Driven by
electron transport)
3. Photophosphorylation (Photosynthesis)
• 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 1,3BPG requires 3phosphoglycerate.
Reaction 8: Phosphoglycerate Mutase
• Repositions the phosphate
• Mutase: catalyzes migration of a functional
group within a substrate
• Phosphoenzyme intermediates
• A bit of 2,3-BPG is required as a cofactor
The catalytic His183 at the active site of E. coli phosphoglycerate
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
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
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.
Large, negative DG -- 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
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
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.
Pyruvate also has two possible fates:
1. aerobic: into citric acid cycle (chapter
19) where it is oxidized to CO2 with the
production of additional NADH (and
2. anaerobic: (fermentation)
In yeast: reduced to ethanol
• Pyruvate decarboxylase (TPP)
• Alcohol dehydrogenase (Reoxidized
– In animals: reduced to lactate
• Lactate dehydrogenase (Reoxidized NADH
to NAD+)
18.6 – How Do Cells Regulate Glycolysis?
The elegant evidence of regulation (See
Figure 18.22)
• Standard state DG values are variously
positive and negative
DG in cells is revealing:
– Most values near zero (reactions 2 and 4-9)
– 3 of 10 Reactions have large, negative DG
• Large negative DG Reactions are sites of
1. Hexokinase
2. Phosphofructokinase
3. Pyruvate kinase
Overview of the regulation
of glycolysis.
18.7 – Are Substrates Other Than Glucose
Used in Glycolysis?
Sugars other than glucose
can be glycolytic
• Fructose and mannose
are routed into
glycolysis by fairly
conventional means.
 Fructose
In liver
1. Fructokinase
Fructose + ATP  fructose-1-phosphate + ADP + H+
2. Fructose-1-phosphate aldolase
fructose-1-phosphate  glyceraldehyde + DHAP
3. Triose kinase
glyceraldehyde  glyceraldehyde-3-phosphate
In kidney and muscle
• Hexokinase
Fructose + ATP  fructose-6-phosphate + ADP + H+
 Mannose
1. Hexokinase
mannose + ATP  mannose-6-phosphate + ADP + H+
2. Phosphomannoisomerase
mannose-6-phosphate  fructose-6-phosphate
 Galactose is more interesting - the Leloir
pathway "converts" galactose to glucose
1. Galactokinase
Galactose + ATP  galactose-1-phosphate + ADP + H+
2. Galactose-1-phosphate uridylyltransferase
3. Phosphoglucomutase
4. UDP-galactose-4-epimerase
Figure 18.24 Galactose
metabolism via the Leloir
Figure 18.25 The galactose-1-phosphate uridylyltransferase
reaction involves a “ping-pong” kinetic mechanism.
─ 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
Lactose Intolerance
• The absence of the enzyme lactase (b-galactosidase)
• Diarrhea and discomfort
• 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
18.8 How Do Cells Respond to Hypoxic
Glycolysis is an anaerobic pathway—it does
not require oxygen
1.The TCA (tricarboxylic acid) cycle is
aerobic. When oxygen is abundant, cells
prefer to combine these pathways in aerobic
2.When oxygen is limiting, cells adapt to
carry out more glycolysis
• 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
• 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)
Hypoxia inducible factor (HIF)
• A heterodimer consists of two subunits:
1. A constitutive, stable nuclear b subunit HIF-1β
2. An inducible, unstable hypoxia-responsive HIF-α
•Bind to the hypoxia responsive element (HRE) of
hypoxia-inducible genes—Activating transcription of
these genes
• HIF-α regulation is a multistep process
–Gene splicing
–Acetylation (Inhibited)
–Hydroxylation (Inhibited)
–Phosphorylation (activated)
• Under normal oxygen levels, HIF-a are
synthesized but quickly degraded
• When oxygen is plentiful, HIF-1a is
hydroxylated by the prolyl hydroxylases
– 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
– FIH-1 (hydroxylase factor-inhibiting HIF-a)
hydroxylates HIF-a at Asn803
• PHDs and FIH-1 both are oxygen-dependent
Figure 18.28