Transcript Lecture 24

Lecture 24
– Quiz Mon. on Pentose Phosphate Pathway
– Glycogen regulation
– Quiz next Fri. on TCA cycle
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Figure 23-25 The pentose phosphate pathway.
Glycogen biosynthesis
Most important storage form of sugar
Glycogen - highly branched (1 per 10) polymer of glucose
with (1,4) backbone and (1,6) branch points. More
branched than starch so more free ends.
Average molecular weight -several million in liver, muscle.
1/3 in liver (more concentrated but less overall mass (5-8%)),
2/3 in muscle (1%).
Not found in brain - brain requires free glucose (120 g/ day)
supplied in diet or from breakdown of glycogen in the liver.
Glucose levels regulated by several key hormones - insulin,
glucagon.
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Figure 18-1a Structure of glycogen. (a)
Molecular formula.
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Figure 18-1bStructure of glycogen.
(b) Schematic diagram illustrating its
branched structure.
Glycogen is an efficient storage form
UDP-glucose
G-6-P
G-1-P + UTP
UDP + ATP
Glycogen + UDP + Pi
UTP + ADP
Net: 1 ATP required
90% 1,4 residues Glycogen + Pi
10% 1,6 residues
Glycogen
G-1-P
G-6-P
glucose
1.1 ATP/38 ATP so, about a 3% loss, therefore it is about 97%
efficient for storage of glucose
Glycogen biosynthesis
3 enzymes catalyze the steps involved in glycogen
synthesis:
UDP-glucose pyrophosphorylase
Glycogen synthase
Glycogen branching enzyme
Glycogen biosynthesis
MgATP
Glucose
HK
MgADP
[G-1,6-P2]
G-6-P
F-6-P
G-1-P
phosphoglucomutase
PGI
The hydrolysis of
pyrophosphate to
inorganic phosphate
is highly exergonic
and is catalyzed by
inorganic
pyrophosphatase
PPase
UTP
G-1-P
PPi
UDP-Glucose Pyrophosphorylase
2Pi
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Figure 18-6 Reaction catalyzed by UDP–glucose
pyrophosphorylase.
UDP-Glucose pyrophosphorylase
Coupling the highly exergonic cleavage of a nucleoside
triphosphate to form PPi is a common biosynthetic strategy.
The free energy of the hydrolysis of PPi with the NTP
hydrolysis drives the reaction forward.
Glycogen synthase
In this step, the glucosyl unit of UDP-glucose (UDPG) is
transferred to the C4-OH group of one of glycogen’s
nonreducing ends to form an (1,4) glycosidic bond.
Involves an oxonium ion intermediate (half-chair
intermediate)
Each molecule of G1P added to glycogen regenerated
needs one molecule of UTP hydrolyzed to UDP and Pi.
UTP is replenished by nucleoside diphosphate kinase
UDP + ATP
UTP + ADP
Figure 18-7 Reaction catalyzed by glycogen synthase.
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O
Glycogen synthase
All carbohydrate biosynthesis occurs via UDP-sugars
Can only extend an already (1,4) linked glucan change.
First step is mediated by glycogenin, where glucose is
attached to Tyr 194OH group.
The protein dissociates after glycogen reaches a minimum
size.
Glycogen branching
Catalyzed by amylo (1,41,6)-transglycosylase (branching
enzyme)
Branches are created by the terminal chain segments
consisting of 7 glycosyl residues to the C6-OH groups of
glucose residues on another chain.
Each transferred segment must be at least 11 residues.
Each new branch point at least 4 residues away from other
branch points.
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Figure 18-8
The branching of glycogen.
Glycogen Breakdown
Requires 3 enzymes:
1. Glycogen phosphorylase (phosphorylase) catalyzes
glycogen phosphorylysis (bond cleavage by the
substitution of a phosphate group) and yields glucose-1phosphate (G1P)
2. Glycogen debranching enzyme removes glycogen’s
branches, allowing glycogen phosphorylase to complete
it’s reactions. It also hydrolyzes a(16)-linked glucosyl units
to yield glucose. 92% of glycogen’s glucse residues are
converted to G1P and 8% to glucose.
3. Phosphoglucomutase converts G1P to G6P-can either
go through glycolysis (muscle cells) or converted to
glucose (liver).
Glycogen Phosphorylase
A dimer - 2 identical 842 residue subunits.
Catalyzes the controlling step of glycogen breakdown.
Regulated by allosteric interactions and covalent modification.
Two forms of phosphorylase made by regulation
Phosphorylase a- has a phosphoryl group on Ser14 in each
subunit.
Phosphorylase b-lacks the phosphoryl groups.
Inhibitors: ATP, G6P, glucose
Activator: AMP
Glycogen forms a left-handed helix with 6.5 glucose residues
per turn.
Structure can accommodate 4-5 sugar residues only.
Pyridoxal phosphate is an essential cofactor for
phosphorylase.
Converts glucosyl units of glycogen to G1P
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Figure 18-2a X-Ray structure of rabbit muscle glycogen
phosphorylase. (a) Ribbon diagram of a phosphorylase
b subunit.
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Phosphoglucomutase
Converts G1P to G6P.
Reaction is similar to that of phosphoglycerate mutase
Difference between phosphoglycerate mutase and
phosphoglucomutase is the amino acid residue to which
the phosphoryl group is attached.
Serine in phosphoglucomutase as opposed to His imidazole
N in phosphoglycerate mutase.
G1,6P occasionally dissociates from the enzyme, so catalytic
amounts are necessary for activity. This is supplied by the
enzyme phosphoglucokinase.
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Figure 18-4 The mechanism of action of
phosphoglucomutase.
Glycogen debranching enzyme
(14) transglycosylase (glycosyl transferase) transfers a
(14) linked trisaccharide unit from a limit branch to a
nonreducing end of another branch.
Forms a new (14) linkage with three more units available
for phosphorylase.
The (16) bond linking the remaining linkage is hydrolyzed
by the same enzyme to yield glucose.
2 active sites on the same enzyme.
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Figure 18-5 Reactions catalyzed by debranching
enzyme.
Regulation of glycogen synthesis
Both synthase & phosphorylase exist in two forms.
Phosphorylated at Ser residues by synthase kinase and
phosphorylase kinase
Synthase a
Normal form
“active”
Pi
OH
OH
phosphoprotein
phosphatase
ATP
Synthase kinase
ADP
Synthase b
Requires G6P for activation
“inactive”
OP
OP
Regulation of glycogen synthesis
AMP+, ATP-, G6PPhosphorylase b
Normal form
“inactive”
Pi
phosphoprotein
phosphatase
OH
OH
ATP
phosphorylase
kinase
Ca2+
ADP
Phosphorylase a
OP
Independent of energy status
OP
active
High [ATP] (related to high G6P) inhibits phosphorylase
and stimulates glycogen synthase.
Regulation of glycogen synthesis
Process is also under hormonal control
Adrenaline (epinephrine) can regulate glycogen
synthesis/breakdown by stimulating adenylate cyclase
ATP
1. External stimulus
Adrenaline
Adenylate
cAMP
cyclase
2. R2C2
cAMP dependent
protein kinase
“inactive”
[C]2 + [R-AMP]2
“active”
ATP
ADP
Glycogen synthase b (inactive)
3a. Glycogen synthase a
[C]2
(active)
ATP
3b. Inactive
phosphorylase kinase
Phosphorylase b
(inactive)
cAMP
+ PPi
ADP
Active phosphorylase kinase
[C]2
ATP
ADP
Phosphorylase a
(active)
Consider the whole system
Resting muscle
Glycolytic pathway
pyruvate
O2
respiration
ATP
Inactive phosphorylase b, active synthase a
Muscle lacks G6 Pase, Liver PFK inhibited by ATP unless F2,6P2 present
Upon stress
Epinephrine
cAMP
Phosphorylse b
Synthase/phosphorylase kinase
Phosphorylse a
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Figure 23-25 The pentose phosphate pathway.
Why is the pentose phosphate pathway
necessary?
• ATP is the “energy currency” of cells, but cells also
need reducing power.
• Endergonic reactions require reducing power and
ATP
– Fatty acids, cholesterol, photosynthesis
• NADPH and NADH are not interchangeable!
– Differ only by a phosphate group at the 2’OH.
Common carrier of (H)
O
N
N
O
N
N
O
HO
C-N-H2
O
CH2-O-P-O-P-CH2
O
O- OOH
HO
N(+)
OH
Pi
NAD(P) Nicotinamide adenine dinculeotide (phosphate)
(oxidized form)
NAD+ + 2e-
NADH + H+
Common carrier of (H)
H
H
N
N
O
N
N
O
HO
C-N-H2
O
CH2-O-P-O-P-CH2
O
O- OOH
O
HO
N
OH
Pi
NAD(P) Nicotinamide adenine dinculeotide (phosphate)
(reduced form)
NADH + H+
NAD+ + 2e-
Eº ‘ = 0.31 volt
Pentose phosphate pathway
•
•
•
•
•
•
•
NADPH and NADH are not interchangeable!
– Differ only by a phosphate group at the 2’OH.
NADH participates in utilizing the free energy of metabolite oxidation to
synthesize ATP
NADPH utilizes the free energy of metaboite oxidation for biosynthesis
Difference is possible because the dehydrogenase enzymes involved in
oxidative and reductive metabolism exhibit a high degree of specificity
toward their respective coenzymes.
Ratios different:
[NAD+]/[NADH] is near 1000 which favors metabolite oxidation.
[NADP+]/[NADPH] is near 0.1 which favors metabolite reduction.
Why is the pentose phosphate pathway
necessary?
• NADPH is generated by oxidation of G6P via the pentose
phosphate pathway
– hexose monophosphate (HMP) pathway, phosphogluconate pathway.
• Alternate to glycolysis.
• Produces ribose-5-phosphate (essential for nucleotide
biosynthesis).
Overall reaction
3G6P + 6NADP+ + 3H2O
Can be considered in 3 stages
6NADPH + 6H+ + 3CO2
+ 2F6P + GP
Pentose phosphate pathway
Can be divided into three stages
1. Oxidative reactions (1-3) which yield NADPH and ribulose-5phosphate (Ru5P).
3G6P + 6NADP+ + 3H2O
6NADPH + 6H+ + 3CO2
+ 3Ru5P
2. Isomeraization and epimeraztion reactions (4,5)-transform
Ru5P to ribose-5-phosphate (R5P) or to xyulose-5phosphate (Xu5P).
3Ru5P
R5P + 2Xu5P
3. C-C bond cleavage and formation reactions (6-8)-convert
2Xu5P and R5P to 2F6P and GAP
R5P + 2Xu5P
2F6P + GAP
Oxidative reactions of NADPH production (1-3)
1.
2.
3.
Glucose-6-phosphate dehydrogenase (G6PD)-catalyzes
the net transfer of a hydride ion to NADP+ from C1 of G6P
to form 6-phophoglucono--lactone.
6-phosphoglucolactonase-increases the rate of
hydrolysis of 6-phophoglucono--lactone to 6phosphogluconate.
6-phosphogluconate dehydrogenase catalyzes the
oxidative decarboxylation of 6-phosphogluconate, a hydroxy acid, Ru5P and CO2. (similar to isocitrate
dehydrogenase)
Reaction 1: The glucose-6-phosphate
dehydrogenase reaction.
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G6PD is strongly inhibited by NADPH
Reaction 2: 6-phosphoglucolactonase
-2O P-O
3
6-phosphoglucono--lactone
6
O
5
OH
4
1
2
HO
O
3
OH
H2O
O
O
C
H-C-OH
6-phosphoglucolactonase
Mg2+
Spontaneous reaction
sped up by the enzyme
HO-C-H
H-C-OH
H-C-OH
6-phosphogluconate
CH2-OPO32-
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Reaction 3: The phosphogluconate dehydrogenase
reaction.
Summary of 1st stage
• 3 reactions take G6P to Ru5P
– G6P + NADP+
6-phosphoglucono--lactone + NADPH
– 6-phosphoglucono--lactone
6-phosphogluconate
– 6-phosphogluconate + NADP+
Ru5P + CO2 + NADPH
• Generates 2 molecules of NADPH for each G6P
• Ru5P must be converted to R5P or Xu5P for further
use.
2nd stage: isomerization and epimerization
• Ru5P is converted to ribose-5-phosphate (R5P)
by ribulose-5-phosphate isomerase
• Ru5P is converted to xyulose-5-phosphate
(Xu5P) by ribulose-5-phosphate epimerase
• Occur via enediolate intermediates.
• R5P is an essential precursor for nucleotide
biosynthesis.
• If more R5P is formed than the cell needs,
converted to F6P and GAP for glycolysis.
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NADH
ATP
DNA
RNA
3rd stage: carbon-carbon bond cleavage and
formation reactions
• Conversion of three C5 sugars to two C6 sugars and
one C3 (GAP)
• Catalyzed by two enzymes, transaldolase and
transketolase
• Mechanisms generate a stabilized carbanion which
interacts with the electrophilic aldehyde center
Transketolase
•
•
•
1.
2.
3.
4.
Transketolase catalyzes the transfer of C2 unit from Xu5P to R5P
resulting in GAP and sedoheptulose-7-phosphate (S7P).
Reaction involves a covalent adduct intermediate between Xu5P and
TPP.
Has a thiamine pyrophosphate cofactor that stabilizes the carbanion
formed on cleavage of the C2-C3 bond of Xu5P.
The TPP ylid attacks the carbonyl group of Xu5P (C2)
C2-C3 bond cleavage results in GAP and enzyme bound 2-(1,2dihydroxyethyl)-TPP (resonance stabilized carbanion)
The C2 carbanion attacks the aldehyde carbon of R5P forming an
S7P-TPP adduct.
TPP is eliminated yielding S7P and the regenerated enzyme.
Thiamine Pyrophosphate (B1)
very acidic H since
the electrons can
delocalize into
heteroatoms.
H
CH2
N
CH3
N
+
N
S
CH3
CH2CH2O-P-P
Thiazolium ring
Involved in both oxidative and non-oxidative
decarboxylation as a carrier of "active" aldehydes.
Covalent adduct
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Carbanion intermediate
Transketolase
•
•
•
Similar to pyruvate decarboxylase mechanism.
Septulose-7-phosphate (S7P) is the the substrate
for transaldolase.
In a second reaction, a C2 unit is transferred from
a second molecule of Xu5P to E4P (product of
transaldolase reaction) to form a molecule of F6P
Transaldolase
•
•
•
•
•
Transfers a C3 unit from S7P to GAP yielding erythrose-4phosphate (E4P) and F6P.
Reactions occurs by aldol cleavage.
S7P forms a Schiff base with an -amino group of Lys from
the enzyme and carbonyl group of S7P.
Transaldolase and Class I aldolase share a common
reaction mechanism.
Both enzymes are  barrel proteins but differ in where
the Lys that forms the Schiff base is located.
•
Essential Lys residue forms a Schiff
base with S7P carbonyl group
•
A Schiff base-stabilized C3 carbanion
is formed in aldol cleavage reaction
between C3-C4 yielding E4P.
The enzyme-bound resonancestabilized carbanion adds to the
carbonyl C of GAP to form F6P.
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•
•
The Schiff base hydrolyzes to
regenerate the original enzyme and
release F6P
Page 867
Figure 23-31 Summary of carbon skeleton
rearrangements in the pentose phosphate
pathway.
Control of Pentose Phosphate Pathway
1. Principle products are R5P and NADPH.
2. Transaldolase and transketolase convert excess R5P
into glycolytic intermediates when NADPH needs are
higher than the need for nucleotide biosynthesis.
3. GAP and F6P can be consumed through glycolysis and
oxidative phosphorylation.
4. Can also be used for gluconeogenesis to form G6P
5. 1 molecule of G6P can be converted via 6 cycles of
PPP and gluconeogenesis to 6 CO2 molecules and
generate 12 NADPH molecules.
6. Flux through PPP (rate of NADPH production) is
controlled by the glucose-6-phosphate dehydrogense
reaction.
7. G6PDH catalyzes the first committed step of the PPP.