Transcript Glycolysis and the Catabolism of Hexoses

Chapter 15
Glycolysis and
the Catabolism of
Hexoses
An overview on D-glucose metabolism
• The major fuel of most organisms, G'o = –2840 kJ/mole if
completely oxidized to CO2 and H2O via the glycolysis
pathway, citric acid cycle and oxidative phosphorylation
(generating ATP) .
• Can also be oxidized to make NADPH and ribose-5-P via
the pentose phosphate pathway.
• Can be stored in polymer form (glycogen or starch) or be
converted to fat for long term storage.
• Is also a versatile precursor for carbon skeletons of almost
all kinds of biomolecules, including amino acids,
nucleotides, fatty acids, coenzymes and other metabolic
intermediates.
1. The Development of Biochemistry
and the Delineation of Glycolysis
Went Hand by Hand
• 1897, Eduard Buchner (Germany), accidental
observation : sucrose (as a preservative) was rapidly
fermented into alcohol by cell-free yeast extract.
• The accepted view that fermentation is inextricably
tied to living cells (i.e., the vitalistic dogma) was
shaken and Biochemistry was born: Metabolism
became chemistry!
• 1900s, Arthur Harden and William Young Pi is
needed for yeast juice to ferment glucose, a hexose
diphosphate (fructose 1,6-bisphosphate) was isolated.
• 1900s, Arthur Harden and William Young (Great
Britain) separated the yeast juice into two fractions:
one heat-labile, nondialyzable zymase (enzymes)
and the other heat-stable, dialyzable cozymase
(metal ions, ATP, ADP, NAD+).
• 1910s-1930s, Gustav Embden and Otto Meyerhof
(Germany), studied muscle and its extracts:
– Reconstructed all the transformation steps from
glycogen to lactic acid in vitro; revealed that many
reactions of lactic acid (muscle) and alcohol (yeast)
fermentations were the same!
– Discovered that lactic acid is reconverted to
carbohydrate in the presence of O2 (gluconeogenesis);
observed that some phosphorylated compounds are
energy-rich.
• (Glycolysis was also known as EmbdenMeyerhof pathway).
• The whole pathway of glycolysis (Glucose to
pyruvate) was elucidated by the 1940s.
2. The overall glycolytic pathway can
be divided into two phases
• The hexose is first phophorylated (thus activated)
and then cleaved to produce two three-carbon
intermediates at the preparatory phase, consuming
ATP.
• The three-carbon intermediates are then oxidized
during the payoff phase, generating ATP and NADH.
• All intermediates are phosphorylated (as esters or
anhydrides) with six (derivatives of Glucose or
Fructose) or three carbons (derivatives of
dihydroxyacetone, glyceraldehyde, glycerate, or
pyruvate).
• Six types of reactions occur: group transfer
(kinase), isomerization (isomerase), aldol cleavage
(aldolase), dehydrogenation (dehydrogenase),
group shift (mutase), dehydration (dehydratase or
enolase).
• Ten steps of reactions are involved in the pathway.
• Only a small fraction (~5%) of the potential energy
of the glucose molecule is released and much still
remain in the final product of glycolysis, pyruvate.
• All the enzymes are found in the cytosol (pyruvate
will enter mitochondria for further oxidation).
Group
transfer
Isomerization
Group
transfer
Aldol
cleavage
Isomerization
The preparatory
Phase of glycolysis
Dehydrogenation
Group
transfer
Group shift
Dehydration
Group
transfer
The payoff phase of
glycolysis
3. Ten enzymes catalyze the ten
reactions of glycolysis
• Hexokinase (also glucokinase in liver) catalyzes the first
phosphorylation reaction on the pathway: Mg2+ATP2-, not
ATP4- is the actual substrate; binding of glucose induces a
profound conformational change on the enzyme; the
reaction is exergonic and thus thermodynamically
favorable (under standard conditions!).
• Phosphohexose isomerase (also called phosphoglucose
isomerase) catalyzes the isomerization from glucose 6-P
to fructose 6-P, converting an aldose to a ketose.
• Phosphofructokinase-1 (PFK-1, 磷酸果糖激酶-1) then
catalyzes the second phosphorylation step, converting
fructose 6-P to fructose 1,6-bisphosphate; the overall rate
of glycolysis is mainly controlled at this step; PFK-1 is a
highly regulatory enzyme; the plant PFK-1 makes use of
PPi, instead of ATP at this step.
• Aldolase (醛缩酶), named for the reverse reaction
catalyzes the cleavage (“lysis”) of fructose 1,6bisphosphate from the middle C-C bond to form two 3carbon sugars, dihydroxyacetone phosphate and
glyceraldehyde 3-phosphate; this is a reversal aldol
condensation reaction; thermodynamically very
unfavorable under standard conditions.
• Triose phosphate isomerase (an extremely efficient
enzyme) converts dihydroacetone phosphate to
glyceraldehyde 3-phosphate; an intramolecular
redox reaction (a hydrogen atom is transferred from
C-1 to C-3).
• Glyceraldehyde 3-phosphate dehydrogenase
catalyzes first the oxidation and then the
phosphorylation of glyceraldehyde 3-P to form
glycerate 1,3-bisphosphate, an acyl phosphate (酰基
磷酸); 2e- are collected by NAD+; a thioester (硫酯)
intermediate is formed between glyceraldehyde 3-P
and an essential Cys residue of the enzyme; Pi is used
here for the phosphorolysis (磷酸解作用); the
phosphate group linked to the carboxyl group via a
anhydride bond has a high transfer potential.
• The phosphoglycerate kinase catalyzes the direct transfer
of the anhydride phosphate in 1,3-BPG to an ADP to
generate an ATP; this is called the substrate-level
phosphorylation; 1,3-BPG is a high energy intermediate
that leads to ATP formation.
• The phosphoglycerate mutase catalyzes the shift of
phosphoryl group on 3-phosphoglycerate from C-3 to C-2;
2,3-bisphosphoglycerate is both a coenzyme for the
mutase and an intermediate for the reaction; a His residue
on the mutase takes phosphoryl group from C-3 of 2,3BPG and adds it to C-2 of 3-phosphoglycerate, thus
forming a phosphorylation cycle; this mutase act in a very
similar way as phosphoglucomutase.
• Enolase (烯醇酶) catalyzes the elimination of a H2O from
2-phosphoglycerate to generate phosphoenolglycerate
(PEP) with the transfer potential of the phosphoryl group
dramatically increased ( G 0` changed from –17.6 to –
61.9 kJ/mol).
• The pyruvate kinase (named for the reverse reaction)
catalyzes the transfer of the phosphoryl group on PEP to
ADP to form another molecule of ATP by “substrate-level
phosphorylation”; enolpyruvate is formed and is quickly
tautomerized to pyruvate (丙酮酸).
• A net gain of two ATP, two NADH, two
pyruvates are resulted when a glucose
molecule is oxidized via the glycolysis
pathway:
Glucose + 2 ADP + 2Pi + 2NAD+
2 pyruvate + 2ATP + 2H2O + 2NADH + 2H+
Irreversible
in cells
Hexokinase
Induced
fit
Glucose
An aldose
An ketose
Reversible
The committing step
Regulatory
ADP
One subunit
of the tetrameric
phosphofructokinase-1
(PFK-1)
An aldehyde
A ketone
1
4
2
5
The “lysis” 3
step
6
Aldol condensation: The combination of two
carbonyl compounds (e.g., an aldehyde and a ketone)
to form an aldol (a b-hydroxyl-carbonyl compound).
A ketose
An aldose
C-1 no longer carries
a large positive charge:
hydride ion leaves readily
Proposed action mechanism
Of glyceraldehyde 3-P
dehydrogenase
Iodoacetate
碘醋酸
Phosphorolysis
（磷酸解作用）
Energy-rich intermediate
(thioester)
Inactive
enzyme
Substrate-level phosphorylation
For ATP generation
Enzyme is named for the reverse reaction
A proposed action
mechanism for
phosphoglycerate
mutase
4. Glycolytic enzymes may form
multienzyme complexes within cells
• When proteins are purified from extracts of broken
cells in diluted solutions, noncovalent interactions
between proteins could be destroyed.
• Kinetic and physical evidences suggest that the
enzymes act to catalyzed the ten reactions of
glycolysis pathway (as enzymes act in other
metabolic pathways) may assemble into
multienzyme complexes, where intermediates are
directly channeled from one enzyme to another,
without entering the aqueous solutions, a
phenomenon called substrate channeling.
Substrate channeling:
growing evidences
seem to indicate the
formation of multienzyme complexes
for enzymes working
in one metabolic
pathway.
5. Fermentation: pyruvate is converted
to lactic acid or ethanol under
anaerobic conditions
• This occurs to regenerate NAD+ for the glycolysis
pathway to continue when O2 lacks.
• Lactic acid fermentation (occurring in very active
skeleton muscle, some bacteria like lactobacilli):
pyruvate is reduced by NADH, catalyzed by lactate
dehydrogenase.
• The lactate produced in muscle can be converted
back to glucose by gluconeogenesis in the liver of
vertebrates (via the Cori cycle).
• Ethanol fermentation (occurring in yeast and other
microorganisms): pyruvate is first decarboxylated
and then reduced by NADH, catalyzed by pyruvate
decarboxylase and alcohol dehydrogenase
respectively.
• Thiamine pyrophosphate (TPP, 硫胺焦磷酸,
derived from vitamin B1) act as the coenzyme of
the decarboxylase: It converts pyruvate to an
“active aldehyde” and facilitates C-C bond
cleavage adjacent to a carbonyl group (also act in
pyruvate and a-ketoglutarate dehydrogenases,
transketonases); the carbon between the N and S in
the thiazole ring is reactive, where a carbanion (负
碳离子) can be easily generated.
Pyruvate is reduced
to lactate when O2
lacks in a reaction
catalyzed by lactate
dehydrogenase
Named for the
Reverse reaction
Pyruvate can be
decarboxylated
and reduced to
form ethanol
in some microorganisms
Present only in those
alcohol fermentative
organisms
Present in many
organisms including
human
Thiamine pyrophosphate (TPP)
contains a reactive thiazole ring
where a carbanion can be formed
Pyruvate is
decarboyxlated
With the help of
TPP, a coenzyme
of pyruvate
decarboxylase during
ethanol fermentation
6. Glycogen in cells is first converted to
Glc-6-P for oxidative degradation
• The glucose unit at the nonreducing terminal of
glycogen is removed as Glc-1-P via phosphorolysis:
The (a1 4) glycosidic bond is attacked by an
inorganic phosphate).
• Catalyzed by glycogen phosphorylase (a tetramer),
its coenzyme pyridoxal phosphate (PLP, 磷酸吡哆
醛) derived from vitamin B6) act as a general acidbase catalyst.
• Phosphorylase stops working when reaching a
terminal residue four away from a branch point.
• A bifunctional debranching enzyme (160 kD) removes the
(a1 6 ) branches in glycogen: the transferase shifts a
block of three glucosyl residues from one outer branch to
the other; then the (a1 6) glucosidase activity removes the
glucose at the end.
• Glc-1-P is then converted to Glc-6-P by the catalysis of
phosphoglucomutase,（葡萄糖磷酸变位酶) which uses
glucose 1,6-bisphosphate as both a cofactor and an
intermediate.
• An Ser residue on the enzyme facilitates the
phosphorylation cycle (a similar role played by a His
residue in the phosphoglycerate mutase).
• Glc-6-P is further degraded via the glycolysis pathway (or
converted to glucose in liver).
No ATP
Consumed!
Tetrameric
glycogen
phosphorylase
(the b form)
No escape
Pyridoxal
phosphate
AMP
(allosteric
Activator)
PLP acts as a general
acid-base in the active
site of glycogen
phosphorylase
PLP
A bifunctional debranching
enzyme aids the phophorylase
in degrading glycogen.
Glucose 1,6-bisphosphate
Ser
The phosphglucomutase shifts the phosphoryl group
from position C-1 to position C-6 on the glucose unit.
7. Other hexoses are also oxidized via
the glycolysis pathway
• They are also first primed by phosphorylation (at
C-1 or C-6).
• Fructose is primed and cleaved to form
dihydroxyacetone phosphate and glyceraldehyde,
which are further converted to glyceraldehyde 3-P.
• Galactose is first converted to Glc-1-P via a UDPgalactose intermediate and UDP-glucose
intermediate, then to Glc-6-P.
One fructose is converted to
two glyceraldehyde 3-P
Triose phosphate
isomerase
Galactose is converted to
glucose 6-P via a
UDP-galactose intermediate
Glc-P-P-Uridine
8. Dietary poly- and disaccharides are
hydrolyzed to monosaccharides in the
digestive system
• Salivary a-amylase (a-淀粉酶) in the mouth
hydrolyzes starch (glycogen) into short
polysaccharides or oligosacchrides.
• Pancreatic a-amylase (active at low pH) continue
act to convert the saccharides to mainly maltoses
and dextrins (from amylopectin, 枝链淀粉).
• Specific enzymes (e.g., lactase, sucrase, maltase,etc.)
on the microvilli of the intestinal epithelial cells
finally hydrolyze all disaccharides into
monosaccharides.
• The monosacchrides are then absorbed at the
intestinal microvilli and transported to various
tissues for oxidative degradation via the glycolytic
pathway.
• Adults lacking lactase will have lactose
intolerance syndrome: the lactose is converted to
toxic compounds in the large intestine by the
bacteria there, causing abdominal cramps and
diarrhea.
9. Pentose phosphate pathway (戊糖磷
酸途径) converts glucose to specialized
products needed by the cells
• Glc-6-P is first dehydrogenated by a NADP+containing dehydrogenase to form 6phosphoglucono-d-lactone, which is then hydrolyzed
to form 6-phosphogluconate (6-磷酸葡萄糖酸).
• 6-phosphogluconate then undergoes a oxidative
decarboxylation to form D-ribulose 5-P, generating
another molecule of NADPH.
• D-ribulose 5-P is then converted to ribose 5-P.
• When NADPH is the primary requirement in the
cell (as in adipocytes), the pentose phosphates are
recycled into Glc-6-P via a series of
rearrangements of the carbon skeleton, catalyzed
by transketolase ( using TPP) and transaldolase (no
cofactor involved).
• Six five-carbon sugar phosphates are converted to
five six-carbon sugar phosphates.
• Much more active in adipose tissue than in muscle.
• The reverse of this rearrangement, regeneration of
six five-carbon sugar phosphate from five sixcarbon sugar phosphate occurs in the Calvin cycle
(for photosynthetic fixation of CO2 in plants).
The pentose
phosphate
pathway
Looks familiar?
The regeneration of six-carbon
Glucose 6-P from five-carbon
Ribose 5-P in the
Pentose phosphate pathway
核酮糖 5-磷酸
木酮糖 5-磷酸
Ribulose 5-P is first isomerized to form
xylulose 5-P to initiate the regeneration
of glucose 6-P (this reaction is similar to two
reactions in glycolysis, what are they?)
TPP helps the twocarbon transferring
in transketolase
TPP
（转酮醇酶）
Donor
(ketose)
Acceptor
(aldose)
The second reaction catalyzed by transketolase
in converting six ribulose 5-P to five Glc 6-P.
TPP
（转酮醇酶）
Donor
(ketose)
Acceptor
(aldose)
What is in common between this reaction and that catalyzed
by pyruvate decarboxylase?
A three-carbon unit is transferred from a ketose
to an aldose without being helped by cofactors
景天庚酮糖
赤藓糖
转二羟丙酮酶
Donor
(ketose)
Acceptor
(aldose)
10. The glycogen phosphorylase
isozymes in muscle and liver are
regulated and differently
• The carbohydrate metabolism in muscle and liver
serve different physiological roles: oxidative
degradation to generate ATP for muscle; maintain a
constant blood glucose level for liver (producing
and exporting glucose when in demand and
importing and storing when in excess.
• Two isozymes exist (one in liver and one in
muscle), both in two interconvertible forms: the a
form is phosphorylated and more active; the b form
is dephosphorylated and less active.
• Both phosphorylation and dephosphorylation occur (I.e.,
reversible phosphorylation) and catalyzed by specific
phosphorylase b kinase and phosphorylase a phosphatase
respectively.
• The phosphorylase b kinases in muscle and liver are
controlled by two different hormones, epinephrine (肾上腺
素) and glucagon(胰增血糖素) respectively.
• High level of AMP binds to and activates the b form of the
muscle isozyme, which is blocked by a high level of ATP.
• High level of glucose binds to the a form of the liver
isozyme, exposing the phosphorylated Ser residues to the
action of phosphorylase a phosphatase and converting it to
the less active b form.
The liver glycogen
phosphorylase is
regulated by
allosteric effector
AMP in addition to
reversible
phosphorylation
The a form
The b form
AMP is a
positive
regulator
Glucose
PLP
Glycogen
The
a
form
of
Phosphorylase
glycogen
phosphorylase
a (phosphorylated)
AMP
Ser14-P
The reversible
phosphorylations
of the glycogen
phosphorylase
isozymes in liver
and muscle
are regulated by
Different hormones
The muscle glycogen
phosphorylase is negatively
regulated by glucose
11. The rate of glycolysis in mammals
is mainly controlled at the step acted by
phosphofructokinase-1 (PFK-1)
• PFK-1 catalyzes an irreversible exergonic reaction,
which commits glucose to the glycolysis pathway
(away from the pentose phosphate pathway).
• PFK-1 is a complex tetrameric enzyme regulated by
multiple intracellular signals (allosteric effectors):
ATP, citrate being negative ones; AMP, ADP and
fructose 2,6-bisphosphate as positive ones.
• A regulated bifunctional enzyme (PFK-2 and
FBPase-2) synthesizes (from Fru-6-P) and degrades
fructose 2,6-bisphosphate.
• A feedforward stimulation: Fru-6-P stimulate the
synthesis and inhibits the hydrolysis of Fru-2,6bisphosphate, which in turn stimulates PFK-1.
Phosphofructokinase-1
(PFK-1) is regulated by
many negative and
positive effectors
ADP
Fructose 1,6bisphosphate
12. Hexokinase and pyruvate kinase
also set the pace of glycolysis
• These two enzymes also catalyzed irreversible
exergonic reactions.
• Muscle hexokinase is allosterically inhibited by
its reaction product Glc-6-P, which accumulates
when PFK-1 is inhibited.
• The liver hexokinase (also called hexokinase D or
glucokinase) has about 100 X less affinity for
glucose than that in muscle and is not inhibited
by Glc-6-P: its main role is to convert excess
glucose to Glc-6-P for glycogen synthesis.
• Pyruvate kinase is allosterically inhibited by
ATP,alanine, acetyl-CoA, and long-chain fatty
acids.
• The catalytic activity of the liver pyruvate kinase
isozyme (the L type) is also controlled by
reversible phosphorylation.
13. Glycolysis and gluconeogenesis are
coordinately regulated to avoid the
wasteful “futile cycling”
• Gluconeogenesis: The pathway converting simpler
precursors (e.g., pyruvate and lactate) to glucose,
mainly occurring in the liver of mammals.
• Gluconeogenesis uses most of the same enzymes of
glycolysis, but the three exergonic irreversible
reactions (catalyzed by the three regulatory enzymes)
are detoured (bypassed).
• The unique enzymes catalyzing the two reversing
reactions at one detouring step are reciprocally
regulated by common allosteric effectors: fructose
2,6-bisphosphate activates PFK-1 (thus activate
glycolysis) and at the same time inhibits fructose
bisphosphotase 1 or FBPase-1 (thus inhibit
gluconeogenesis).
• Enzymes catalyzing the non-common steps of paired
catabolic and anabolic pathways are often
reciprocally regulated to avoid futile cycling.
Summary
• D-glucose is a commonly used fuel and versatile precursor
in almost all organisms.
• The study of glucose degradation has a rich history in
biochemistry (especially for enzymology).
• Glucose is first converted into two three-carbon pyruvates
via the ten-step glycolysis pathway without directly
consuming O2 and with a net production of two ATP
molecules by substrate-level phosphorylation.
• Limited amount of energy can be released by oxidizing
glucose under anaerobic conditions by fermentation.
• The enzymes participating glycolysis may form
multiple enzyme complexes, where substrate is
channeled from one enzyme to another.
• The sugar units on glycogen is converted to glucose
1-phosphate via phosphorolysis, which is catalyzed
by glycogen phosphorylase.
• Other monosaccharides are also converted to
intermediates of glycolysis for further oxidative
degradation.
• Glucose 6-phosphate can also be oxidized to form
ribose 5-phosphate and NADPH via the pentose
phosphate pathway.
• Glycogen phosphorylase is regulated by allosteric effectors
and reversible phosphorylation, which is in turn controlled
by hormones.
• The liver and muscle glycogen phosphorylases are regulated
different to meet their physiological roles in mammals.
• Phosphofructokinase-1 (PFK-1) is the main point of
regulation for controlling the rate of glycolysis.
• The activity of PFK-1 is regulated by various effectors
having various signaling messages of the cell metabolism.
• Glycolysis and gluconeogenesis is reciprocally regulated to
avoid “futile cycling” of synthesis and degradation.
References
• Suarez, R. K., Staples, J.F., Lighton, J. R., and West, T. G.
(1997) “Relationships between enzymatic flux
capacities and metabolic flux rates: noneequilibrium
reactions in muscle glycolysis” Proc. Natl. Acad. Sci.
USA, 94:7065-7069.
• Barford, D., Hu, S. H., and Jonson, L. N. (1991) “Structural
mechanism for glycogen phosphorylase by
phosphorylation and AMP” J. Mol. Biol., 218:233260.
• Hudson, J. W., Golding, G. B., and Crerar, M. M. (1993)
“Evolution of allosteric control in glycogen
phosphorylase” J. Mol. Biol., 234:700-721.
• Hue, L. and Rider, M. H. (1987) “Role of fructose 2,6bisphosphate in the control of glycolysis in mammalian
tissues” Biochem. J., 245:313-323.
• Schirmer, T and Evans, P. R. (1990) “Structural basis of the
allosteric behavior of phosphofructokinase” Nature, 43:140145.
• Sprang, S. R., Withers, S. G., Goldsmith, E. J., Fletterick, R.
J., and Madsen, N. B. (1991) “Structural basis for the
activation of glycogen phosphorylase b be adenosine
monophosphate” Science, 254:1367-1371.
Glc-1-P can be converted to
UDP-glucuronate and ascorbic acid
Supplementary:
• Via the UDP-Glucose intermediate.
• Consumes NADPH when UDP-glucuronate is
further converted to ascorbic acid (vitamin C).
• Gulonolatone oxidase is a flavoprotein, which is
lacking in human and some other animals
(therefore ascorbic acid can not be made by human
beings).
• UDP-glucuronate is used in glucuronidating
nonpolar toxins, drugs or carcinogens, thus making
them water-soluble and excretable by a family of
detoxifying enzymes.
Glc-1-P can be converted to UDPglucuronate and ascorbic acid
古洛糖酸
葡糖醛酸
古洛糖酸内酯
UDP-glucuronate is
used in detoxification
by glucuronidating
nonpolar toxins.