Download III. Metabolism

Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 3020
III. Metabolism
- Glycolysis
Major Pathways of Glucose Utilization
These three pathways are the most significant in terms of the amount of glucose
that flows through them in most cells.
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The Two Phases of Glycolysis
Breakdown of the six-carbon glucose into two molecules of the
Three-carbon pyruvate occurs in ten steps.
These ten steps can be subdivided in two Phases:
I.
The Preparatory Phase
II.
The Payoff Phase
The Two Phases of Glycolysis
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The Two Phases of Glycolysis
What is the net yield (in energy equivalents) per molecule glucose?
Glucose + 2NAD+ + 2ADP + 2P →
i
+
2 pyruvate + 2NADH + 2H + 2ATP + 2H2O
Formation of 2 Pyruvates:
Glucose + 2NAD+ → 2 pyruvate + 2NADH + 2H+
∆G’1O= -146 kJ/mol
Formation of 2 ATP:
2ADP + 2Pi → 2ATP + 2H2O
∆G’2O= 61.0 kJ/mol
∆G’ O= ∆G’ O + ∆G’ O = -85 kJ/mol
S
1
2
3
A Historical Perspective
•
In years 1854 to 1864, Louis Pasteur established that fermentation is caused
by microorganisms.
•
1897 – Eduard Buchner demonstrated that cell-free yeast extracts can carry
out this process.
•
In years 1905 to 1910, Arthur Harden and William Young discovered:
– Inorganic phosphate is required for fermentation and is incorporated into
fructose-1-6-bisphosphate
– A cell-free yeast extract has a nondialyzable heat-labile fraction (zymase)
and a dialyzable heat-stable fraction (cozymase).
•
Elucidation of complete glycolytic pathway by 1940 (Gustav Embden, Otto
Meyerhof, and Jacob Parnas).
Glycolysis
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Hexokinase: First ATP Utilization
Reaction 1 :
Transfer of a phosphoryl group from ATP to glucose
to form glucose 6-phosphate (G6P)
∆G’° = -16.7 kJ/mol
Hexokinase: First ATP Utilization
In the enzyme-substrate complex the two lobes (grey/green) swing together,
Excluding H20 form the activ site.
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Phosphohexose Isomerase
Recation 2:
Phosphohexose isomerase catalyzes the conversion of G6P
to F6P, essentially the isomerization of an aldose to a ketose.
∆G’° = 1.7 kJ/mol
PFK-1: Second ATP Utilization
Reaction 3:
Phosophofructokinase-1 (PFK-1) phosphorulates
fructose-6-phosphate (F6P)
PFK-1 plays a central role in control of glycolysis
because it catalyzes on of the pathway’s ratedetermening reactions.
∆G’° = -14.2 kJ/mol
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Aldolase
Reaction 4:
Aldolase catalyzes cleavage of fructose-1,6-bisphosphate (FBP)
Breakdown products are: Glyceraldehyde-3-phosphate (GAP)
Dihydroxyaceton phosphate (DHAP)
∆G’° = 23.8 kJ/mol
The Class I Aldolase Reaction
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The Class I Aldolase Reaction Part I
The Class I Aldolase Reaction Part II
8
Triose Phosphate Isomerase
Reaction 5:
Interconversion of the triose phosphates
Only GAP continues along the glycolytic pathway
Dihydroxyacetonphosphate is rapidly and reversible
converted to GAP by TPI
∆G’° = 7.5 kJ/mol
Summary of Reaction 4 & 5
The fate of carbon atoms of glucose in
the formation of glyceraldehyde-3-phosphate
9
Ribbon diagram
of TIM in compex
with its transition
state analog
2-phosphoglycolate.
The flexible loop
(residues 168 -177)
(light blue) makes a
hydrogen bond
with the phosphate
group of the substrate.
Removal of this loop
by mutagenesis does
not impair substrate
binding but reduces
catalytic rate
by 105 fold.
The Payoff Phase
10
Glyceraldehyde-3-Phosphate Dehydrogenase
Reaction 6:
Glyceraldehyde-3-phosphate dehydrogenase forms the
first “high-energy” intermediat.
∆G’° = 6.3 kJ/mol
Glyceraldehyde 3-Phosphate Dehydrogenase
Reaction
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Glyceraldehyde 3-Phosphate Dehydrogenase
Reaction I
Glyceraldehyde 3-Phosphate Dehydrogenase
Reaction II
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Glyceraldehyde 3-Phosphate Dehydrogenase
Reaction III
Glyceraldehyde 3-Phosphate Dehydrogenase
Reaction IV
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Glyceraldehyde 3-Phosphate Dehydrogenase
Reaction V
Phosphoglycerat Kinase: First ATP Generation
∆G’° = -18.5 kJ/mol
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Phosphoglycerat Kinase: First ATP Generation
Upon substrate binding, the
Two domains of PGK swing
Together, providing a waterFree environment
→ hexokinase
3PG
Mechanism of PGK reaction
15
Overall Energetics of the Glyceraldehyde-3Phosphate Dehydrogenase-Phosphoglycerat
Kinase Reaction Pair
Phosphoglycerate Mutase
Reaction 8:
Catalyzes of a reversible shift of the phosphoryl group
2+
between C-2 and C-3 of glycerate; Mg is essential.
∆G’° = 4.4 kJ/mol
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Reaction Mechanism of PGM
Catalytic amounts of 2,3-Bisphosphoglycerate are required for enzymatic activity.
Reaction Mechanism of PGM
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Reaction Mechanism of PGM
Gycolysis Influences Oxygen Transport
2,3-BPG binds to deoxyhemoglobin and alters oxygen affinity.
Erythrocytes synthesize and degrade 2,3-BPG by a detour from the
glycolytic pathway.
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Gycolysis Influences Oxygen Transport
Lower [BPG] in erythrocytes
resulting from hexokinasedeficiency results in
increased hemoglobin
oxygen affinity.
Enolase: Second “High Energy” Intermediate
∆G’° = 7.5 kJ/mol
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Enolase Reaction Mechanism
Enolase Reaction Mechanism
3D structure
Of the catalytic center
(PDB ID 1ONE)
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Pyruvate Kinase : Second ATP Generation
∆G’° = -31.4 kJ/mol
Pyruvate Kinase : Second ATP Generation
Tautomerization of
enolpyruvate to pyruvate.
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Metabolic Fates of NADH and Pyruvate
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Metabolic Fates of NADH and Pyruvate
Pyruvate is a central branch point in
Metabolism.
Aerobic pathway:
Citric acid cycle and respiration;
-Yields far more energy → discussed later
NADH + O2 → NAD+ + Energy
Pyruvate + O2 → 3 CO2 + Energy
Metabolic Fates of NADH and Pyruvate
Pyruvate is a central branch point in
Metabolism.
Two anaerobic pathways:
-Pyruvate is converted to lactate via
lactate dehydrogenase
-Pyrovate is converted to ethanol via
ethanol dehydrogenase
→ Fermentation
Both pathways use up the NADH produced, so only 2 ATP are generated
per glucose consumed.
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Lactate Fermentation
Enzyme = Lactate Dehydrogenase
Pyruvate + NADH + H+
L-Lactate + NAD+
Regenerates NAD+ from NADH (reducing equivalents) produced in glycolysis.
Lactate fermentation is important in red blood cells, parts of the retina and
in skeletal muscle cells during extreme high activity.
Also important in plants and microbes growing in absence of O2.
∆G’° = -25.1 kJ/mol
Lactate Dehydrogenase (LDH)
In mammals two different types of LDH subunits are found:
the M type and the H type.
Five forms of the tetrameric isozymes are possible:
M4, M3H1, M2H2, M1H3, H4
The H-type predominates aerobic tissues such as heart muscle.
The M-type predominates tissue that are subject to anaerobic conditions
such as liver and skeletal muscle.
H4 LDH has a low KM for pyruvate and is allosterically inhibited by it.
M4 LDH has a low KM for pyruvate and is NOT allosterically inhibited by it.
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Lactate Dehydrogenase (LDH)
Reaction Mechanism of Lactate
Dehydrogenase
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Pyruvate is the Terminal Electron Acceptor in
Lactic Acid Fermentation
Much of the lactate, the end product of anaerobic glycolysis,
is exported from the muscle cell via the blood to the liver,
where it is reconverted to glucose.
Alcoholic Fermentation
Two enzymes involved: Pyruvate decarboxylase irreversible
Alcohol dehydrogenase reversible
Regenerates NAD+ from NADH (reducing equivalents) produced in glycolysis.
Pathway is active in yeast
Second step is reversible → ethanol oxidation eventiually yields acetate
→ enters fat synthesis
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Pyruvate Decarboxylase
Yeast produces ethanol and CO2 in two consecutive reactions
The decarboxylation of pyruvate to acetaldehyde is catalyzed by
pyruvate decarboxylase (PDC) (not present in animals).
PDC contains a tightly noncovalently bound coenzyme:
Thiamin pyrophosphate
catalytic active
TPP is an Essential Cofactor of Pyruvate
Decarboxylase
Decarboxylation of α-keto acids
requires the build up of a negative
charge on the carbonyl carbon.
Transition state is stabilized by
delocalization of the developing
neg. charge into a “electron sink”.
The dipolar carbanion (ylid) is the
active form
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TPP is an Essential Cofactor of Pyruvate
Decarboxylase
TPP is an Essential Cofactor of Pyruvate
Decarboxylase
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TPP is an Essential Cofactor of Pyruvate
Decarboxylase
How to deprotonate TPP?
The formation of the active ylid
form of TPP requires the
participation of TTP’s aminopyrimidine
ring together with general acid
catalysis by Glu51 of the second
subunit of the dimer. PDC is a dimer
of dimers.
TPP is an Essential Cofactor of Pyruvate
Decarboxylase
Glu51
29
Thiamine Deficiency
The ability of TTP’s thiazolium ring to add carbonyl groups and act as an
“electron sink” makes it the coenzyme most utilized in α-keto acid
decarboxylations.
Thiamin (vitamin B1) is neither synthesized nor stored in significant amounts by
vertebrates. Deficiency in humans results in an ultimately fatal condition known
as beriberi.
Alcoholic Fermentation Part II
Reduction of Acetaldehyde to Ethanol and Regeneration of NAD+
by alcohol dehydrogenase (ADH)
2+
Each subunit of the tetrameric yeast ADH binds one NADH and one Zn .
30
Alcoholic Fermentation Part II
Zn2+ polarises the carbonyl oxigen
of acetaldehyde
A hydride ion is transferred
The reduced intermediate aquires a
proton from the medium to form
ethanol.
Are Substrates Other Than Glucose Used in
Glycolysis?
Glycogen / Starch
Dietary Polysaccharides
Maltose
Lactose
Sucrose
31
Feeder Pathways for Glycolysis
Glycogen metabolism
Glycogen storage granules
in liver
Glycogen and Starch Are Degraded by
Phosphorolysis
Glycogen phosphorylase /
Starch phosphorylase
-attack of Pi on the (α1→4)
glycosidic linkage of
the last the two glucose
residues.
Phosphorolysis generates
Glucose 1-phosphat
32
Glycogen and Starch Are Degraded by
Phosphorolysis
Phosphorylase acts
repetitively until it
reaches a (α1→6)
A debranching enzyme
is needed
Phosphoglucomutase mechanism
Glucose 1-phosphate
has to be converted into
Glucose 6-phosphate
in order to enter glcolysis
33
Phosphoglucomutase mechanism
Glucose 1-phosphate
has to be converted into
Glucose 6-phosphate
in order to enter glcolysis
Remember that mechanism?
Phosphoglycerate Mutase – Reaction 8
34
But ! – The Liver
Glucose-6-phosphate needs to be converted to glucose for transport to tissue.
Separate from glycolytic pathway!
Dietary Polysaccharides
Dextrin + n H20 → n D-glucose
Dextrinase
Maltose + H20 → 2 D-glucose
Maltase
Lactose + H20 →
D-galactose + D-glucose
Lactase
Sucrose + H20 →
D-fructose + D-glucose
Sucrase
Monosaccharides formed are funneled into the glycolytic sequence
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Entry into the Preparatory stage of Glycolysis
Entry into the Preparatory stage of Glycolysis
D-Fructose is phosphorylated by hexokinase:
Mg
2+
Fructose + ATP → fructose 6-phosphate + ADP
But in liver fructokinase catalyzes the phosphorylation at C1:
Mg
2+
Fructose + ATP → fructose 1-phosphate + ADP
Fructose 1-phosphate is cleaved to glyceraldehyde and
dihydroxyaceton phosphate by fructose 1-phosphate aldolase.
Glyceraldehyde is phosphorylated by triose kinase and ATP to
glyceraldehyde 3-phosphate.
36
Entry into the Preparatory stage of Glycolysis
Conversion of Galactose to Glucose 1phosphate
Metabolism of Galactose involves a
sugar nucletide.
C1 carbon is activated
through phosphate ester
37
Conversion of Galactose to Glucose 1phosphate
Galactose 1-phosphate Uridylyltransferase
38
Conversion of Galactose to Glucose 1phosphate
Conversion of Galactose to Glucose 1phosphate
39
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