Moreover, because glycolysis generates only a fraction of the total energy available from the breakdown of glucose the rest is generated by the TCA cycle and oxidative phosphorylation, t
Trang 1process Pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase in an
essentially irreversible reaction Thiamine pyrophosphate (see page 568) is a required
cofactor for this enzyme The second step, the reduction of acetaldehyde to ethanol
by NADH, is catalyzed by alcohol dehydrogenase (Figure 18.21) At pH 7, the reaction
equilibrium strongly favors ethanol The end products of alcoholic fermentation are
thus ethanol and carbon dioxide Alcoholic fermentations are the basis for the
brew-ing of beers and the fermentation of grape sugar in wine makbrew-ing Lactate produced
by anaerobic microorganisms during lactic acid fermentation is responsible for the
taste of sour milk and for the characteristic taste and fragrance of sauerkraut, which
in reality is fermented cabbage.
Lactate Accumulates Under Anaerobic Conditions in Animal Tissues
In animal tissues experiencing anaerobic conditions, pyruvate is reduced to lactate.
Pyruvate reduction occurs in tissues that normally experience minimal access to
blood flow (for example, the cornea of the eye) and also in rapidly contracting
skeletal muscle When skeletal muscles are exercised strenuously, the available
tis-sue oxygen is consumed and the pyruvate generated by glycolysis can no longer be
oxidized in the TCA cycle Instead, excess pyruvate is reduced to lactate by lactate
dehydrogenase (Figure 18.21) The rate of anaerobic glycolysis in skeletal muscle
can increase up to 2000-fold almost instantaneously, for example, to support the
in-tense demands of a sprinting animal Large amounts of ATP are generated rapidly,
at the expense of lactate accumulation In anaerobic muscle tissue, lactate
repre-sents the end of glycolysis Anyone who exercises to the point of depleting available
muscle oxygen stores knows the cramps and muscle fatigue associated with the
buildup of lactic acid in the muscle Most of this lactate must be carried out of the
muscle by the blood and transported to the liver, where it can be resynthesized into
glucose in gluconeogenesis Moreover, because glycolysis generates only a fraction
of the total energy available from the breakdown of glucose (the rest is generated
by the TCA cycle and oxidative phosphorylation), the onset of anaerobic conditions
in skeletal muscle also means a reduction in the energy available from the
break-down of glucose.
C
+
CHO
CH2OPO3–
HPO4–
C
CH2OPO3–
OPO3– O
H+
CH3CHO
CH3C COO–
O
Pyruvate
CH3CH2OH
H
+
OH CHO
CH2OPO3–
HPO4–
C
CH2OPO3–
OPO3– O
H+
CH3C COO–
COO–
O
CH3 C OH
H
G3PDH
CO2
D
-Glyceraldehyde-3-phosphate
1,3-BPG
Acetaldehyde Ethanol
D -Glyceraldehyde-3-phosphate
1,3-BPG
Lactate
Pyruvate
Lactate dehydrogenase
(a) Alcoholic fermentation (b) Lactic acid fermentation
G3PDH
Alcohol dehydrogenase
FIGURE 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NADconsumed
in the glyceraldehyde-3-P dehydrogenase reaction (b) In oxygen-depleted muscle, NADis regenerated in the
lactate dehydrogenase reaction
Trang 218.6 How Do Cells Regulate Glycolysis?
The elegance of nature’s design for the glycolytic pathway may be appreciated through
an examination of Figure 18.22 The standard-state free energy changes for the 10 re-actions of glycolysis and the lactate dehydrogenase reaction (Figure 18.22a) are vari-ously positive and negative and, taken together, offer little insight into the coupling that occurs in the cellular milieu On the other hand, the values of G under cellular
conditions (Figure 18.22b) fall into two distinct classes For reactions 2 and 4 through
9, G is very close to zero, meaning these reactions operate essentially at equilibrium.
Small changes in the concentrations of reactants and products could “push” any of these reactions either forward or backward By contrast, the hexokinase, phospho-fructokinase, and pyruvate kinase reactions all exhibit large negative G values under
cellular conditions These reactions are thus the sites of glycolytic regulation When these three enzymes are active, glycolysis proceeds and glucose is readily metabolized
to pyruvate or lactate Inhibition of the three key enzymes by allosteric effectors brings
glycolysis to a halt When we consider gluconeogenesis—the biosynthesis of glucose—
in Chapter 22, we will see that different enzymes are used to carry out reactions 1, 3, and 10 in reverse, effecting the net synthesis of glucose The maintenance of reactions
2 and 4 through 9 at or near equilibrium permits these reactions (and their respective
enzymes!) to operate effectively in either the forward or reverse direction.
18.7 Are Substrates Other Than Glucose Used in Glycolysis?
The glycolytic pathway described in this chapter begins with the breakdown of glucose, but other sugars, both simple and complex, can enter the cycle if they can be converted by appropriate enzymes to one of the intermediates of glycoly-sis Figure 18.23 shows the routes by which several simple metabolites can enter
(a) ΔG at standard state (ΔG°')
40
30
20
10
0
–10
–20
–30
–40
0
Steps of glycolysis
1 2 3 4 5 6 7 8 9 10 11
40
30
20
10
0
–10
–20
–30
–40
0
Steps of glycolysis
1 2 3 4 5 6 7 8 9 10 11
(b)ΔG in erythrocytes (ΔG)
FIGURE 18.22 A comparison of free energy changes for
the reactions of glycolysis (step 1 hexokinase) under
(a) standard-state conditions and (b) actual intracellular
conditions in erythrocytes The values of G° provide
little insight into the actual free energy changes that
occur in glycolysis On the other hand, under
intracellu-lar conditions, seven of the glycolytic reactions operate
near equilibrium (with G near zero).The driving force
for glycolysis lies in the hexokinase (1),
phosphofructo-kinase (3), and pyruvate phosphofructo-kinase (10) reactions The lactate
dehydrogenase (step 11) reaction also exhibits a large
negative G under cellular conditions.
G-6-P
F-6-P FBP
2 Pyruvate
Mannose-6-P Mannose
Galactose
Galactose-1-P UDP-Gal
UDP-Glucose
Glucose-1-P
Fructose
Fructose-1-P
D-Glyceraldehyde
Aldolase
Fructokinase
Triose kinase Glucose
3-PG
2-PG
PEP
DHAP
G-3-P
BPG
FIGURE 18.23 Mannose, galactose, fructose, and other simple metabolites can enter the glycolytic pathway
Trang 3the glycolytic pathway Fructose, for example, which is produced by breakdown of
sucrose, may participate in glycolysis by at least two different routes In the liver,
fructose is phosphorylated at C-1 by the enzyme fructokinase:
D-Fructose ATP4⎯⎯→D-fructose-1-phosphate2 ADP3 H (18.9)
Subsequent action by fructose-1-phosphate aldolase cleaves fructose-1-P in a
man-ner like the fructose bisphosphate aldolase reaction to produce dihydroxyacetone
phosphate and D-glyceraldehyde:
D-Fructose-1-P2⎯⎯→D-glyceraldehyde dihydroxyacetone phosphate2 (18.10)
Dihydroxyacetone phosphate is of course an intermediate in glycolysis D
-Glycer-aldehyde can be phosphorylated by triose kinase in the presence of ATP to form
D-glyceraldehyde-3-phosphate, another glycolytic intermediate.
In the kidney and in muscle tissues, fructose is readily phosphorylated by
hex-okinase, which, as pointed out previously, can utilize several different hexose
sub-strates The free energy of hydrolysis of ATP drives the reaction forward:
D-Fructose ATP4⎯⎯→D-fructose-6-phosphate2 ADP3 H (18.11)
HUMAN BIOCHEMISTRY
Tumor Diagnosis Using Positron Emission Tomography (PET)
More than 70 years ago, Otto Warburg at the Kaiser Wilhelm
In-stitute of Biology in Germany demonstrated that most animal and
human tumors displayed a very high rate of glycolysis compared
to that of normal tissue This observation from long ago is the
ba-sis of a modern diagnostic method for tumor detection called
positron emission tomography, or PET PET uses molecular
probes that contain a neutron-deficient, radioactive element such
as carbon-11 or fluorine-18 An example is 2-[18 F]fluoro-2-deoxy-glucose (FDG), a molecular mimic of F]fluoro-2-deoxy-glucose The 18F nucleus is unstable and spontaneously decays by emission of a positron (an antimatter* particle) from a proton, thus converting a proton to
a neutron and transforming the 18F to 18O The emitted positron typically travels a short distance (less than a millimeter) and col-lides with an electron, annihilating both particles and creating a pair of high-energy photons—gamma rays Detection of the gamma rays with special cameras can be used to construct three-dimensional models of the location of the radiolabeled molecu-lar probe in the tissue of interest
FDG is taken up by human cells and converted by hexokinase
to 2-[18F]fluoro-2-deoxy-glucose-6-phosphate in the first step of glycolysis Cells of a human brain, for example, accumulate FDG
in direct proportion to the amount of glycolysis occuring in those cells Tumors can be identified in PET scans as sites of unusually high FDG accumulation
HO OH
18F HOH
(a)
(b)
CH2OH
2-[ 18 F]Fluoro-2-deoxyglucose
Electron in tissue
511 kev Photon
511 kev Photon
18F
18O
Emitted positron
O
e +
e –
(c) PET image of human brain following administration of
18FDG Red area indicates a large malignant tumor
*The existence of antimatter in the form of positrons was first postulated by Robert Oppenheimer, the father of the atomic bomb
Trang 4Fructose-6-phosphate generated in this way enters the glycolytic pathway directly in step 3, the second priming reaction This is the principal means for channeling fructose into glycolysis in adipose tissue, which contains high levels of fructose.
Mannose Enters Glycolysis in Two Steps
Another simple sugar that enters glycolysis at the same point as fructose is
mannose, which occurs in many glycoproteins, glycolipids, and polysaccharides (see Chapter 7) Mannose is also phosphorylated from ATP by hexokinase, and the mannose-6-phosphate thus produced is converted to fructose-6-phosphate by
phosphomannoisomerase.
D-Mannose ATP4⎯ D-mannose-6-phosphate2 ADP3 H (18.12)
D-Mannose-6-phosphate2⎯⎯→D-fructose-6-phosphate2 (18.13)
Galactose Enters Glycolysis Via the Leloir Pathway
A somewhat more complicated route into glycolysis is followed by galactose, an-other simple hexose sugar The process, called the Leloir pathway after Luis
Leloir, its discoverer, begins with phosphorylation from ATP at the C-1 position by
galactokinase:
D-Galactose ATP4⎯⎯→D-galactose-1-phosphate2 ADP3 H (18.14)
Galactose-1-phosphate is then converted into UDP-galactose (a sugar nucleotide)
by galactose-1-phosphate uridylyltransferase (Figure 18.24), with concurrent
pro-duction of glucose-1-phosphate and consumption of a molecule of UDP-glucose The uridylyltransferase reaction (Figure 18.25) proceeds via a “ping-pong” mech-anism (see Chapter 13, page 406) with a covalent enzyme-UMP intermediate The glucose-1-phosphate produced by the transferase reaction is a substrate for the
phosphoglucomutase reaction (Figure 18.24), which produces glucose-6-phosphate, a glycolytic substrate The other transferase product, UDP-galactose, is
converted to UDP-glucose by UDP-glucose-4-epimerase The combined action of
the uridylyltransferase and epimerase thus produces glucose-1-P from galactose-1-P, with regeneration of UDP-glucose.
A rare hereditary condition known as galactosemia involves defects in
galactose-1-P uridylyltransferase that render the enzyme inactive Toxic levels of galactose accumulate in afflicted individuals, causing cataracts and permanent neurological disorders These problems can be prevented by removing galactose and lactose from the diet In adults, the toxicity of galactose appears to be less severe, due in
part to the metabolism of galactose-1-P by UDP-glucose pyrophosphorylase, which
uridylyltransferase
UDP-Galactose-4-epimerase
Phosphoglucomutase
Galactokinase
Galactose
P
Galactose-1-P
UDP-Glucose
UDP-Galactose
P
Glucose-1-P
Glucose-6-ATP
ADP
FIGURE 18.24 Galactose metabolism via the Leloir
pathway
Trang 5apparently can accept galactose-1-P in place of glucose-1-P (Figure 18.26) The
levels of this enzyme may increase in galactosemic individuals in order to
accom-modate the metabolism of galactose.
An Enzyme Deficiency Causes Lactose Intolerance
A much more common metabolic disorder, lactose intolerance, occurs
com-monly in most parts of the world (notable exceptions being some parts of Africa
and northern Europe) Lactose intolerance is an inability to digest lactose
be-cause of the absence of the enzyme lactase in the intestines of adults The
symp-toms of this disorder, which include diarrhea and general discomfort, can be
re-lieved by eliminating milk from the diet.
Glycerol Can Also Enter Glycolysis
Glycerol is the last important simple substance whose ability to enter the glycolytic
pathway must be considered This metabolite, which is produced in substantial
amounts by the decomposition of triacylglycerols (see Chapter 23), can be converted
O
O P
P
O–
HO
CH2OH
O
O P O–
H
CH2OH O O
O–
O P O–
HO
CH2OH
O
O P
P
O–
HO
CH2OH O
+
OH
HO OH
FIGURE 18.25 The galactose-1-phosphate uridylyl-transferase reaction involves a “ping-pong” kinetic mechanism
O–
O P O–
H
CH2OH
O
O
O HN
HO
CH2OH O O
HO
OH
O
OH HO
H
N O
O
O–
O
O–
O
O–
–O
CH2 O
OH HO
H
O
O–
O
O–
HN N O
O
O–
O
O–
+
-D -Galactose-1-P
UDP-galactose (UDP-Gal)
UTP
reaction also works with galactose-1-P
Trang 6to glycerol-3-phosphate by the action of glycerol kinase and then oxidized to dihy-droxyacetone phosphate by the action of glycerol phosphate dehydrogenase, with
NADas the required coenzyme The dihydroxyacetone phosphate thereby produced enters the glycolytic pathway as a substrate for triose phosphate isomerase.
HUMAN BIOCHEMISTRY
Lactose—From Mother’s Milk to Yogurt—and Lactose Intolerance
Lactose is an interesting sugar in many ways In placental
mam-mals, it is synthesized only in the mammary gland, and then only
during late pregnancy and lactation The synthesis is carried out
by lactose synthase, a dimeric complex of two proteins: galactosyl
transferase and -lactalbumin Galactosyl transferase is present in
all human cells, and it is normally involved in incorporation of
galactose into glycoproteins In late pregnancy, the pituitary
gland in the brain releases a protein hormone, prolactin, which
triggers production of -lactalbumin by certain cells in the breast.
-Lactalbumin, a 123-residue protein, associates with galactosyl
transferase to form lactose synthase, which catalyzes the reaction:
UDP-galactose glucose ⎯⎯→ lactose UDP
Lactose breakdown by lactase in the small intestine provides
newborn mammals with essential galactose for many purposes,
including the synthesis of gangliosides in the developing brain
Lactase is a -galactosidase that cleaves lactose to yield galactose
and glucose—in fact, the only human enzyme that can cleave a
-glycosidic linkage:
Lactase is an inducible enzyme in mammals, and it appears in the fetus only during the late stages of gestation Lactase activity peaks shortly after birth, but by the age of 3 to 5 years, it declines to a low level in nearly all human children Low levels of lactase make many
adults lactose intolerant Lactose intolerance occurs commonly in
most parts of the world (with the notable exception of some parts of Africa and northern Europe; see table) The symptoms of lactose in-tolerance, including diarrhea and general discomfort, can be relieved by eliminating milk from the diet Alternatively, products containing-galactosidase are available commercially.
Certain bacteria, including several species of Lactobacillus, thrive
on the lactose in milk and carry out lactic acid fermentation, con-verting lactose to lactate via glycolysis This is the basis of produc-tion of yogurt, which is now popular in the Western world but of Turkish origin Other cultures also produce yogurtlike foods No-madic Tatars in Siberia and Mongolia used camel milk to make
koumiss, which was used for medicinal purposes In the Caucasus, kefir is made much like yogurt, except that the starter culture
con-tains (in addition to Lactobacillus) Streptococcus lactis and yeast,
which convert some of the glucose to ethanol and CO2, producing
an effervescent and slightly intoxicating brew
HO OH OH HOH
CH2OH O HO
OH
OH
CH2OH
O
OH OH HOH
CH2OH O O
HO
OH OH
Lactose
CH2OH O
Lactase
䊱 Breakdown of lactose to galactose and glucose by lactase.
Portions adapted from Hill, R., and Brew, K., 1975 Lactose synthetase
Ad-vances in Enzymology 43:411–485; and Bloch, K., 1994 Blondes in Venetian
Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry New
Haven, CT: Yale University Press
Adapted from Bloch, K., 1994 Blondes in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry New Haven, CT: Yale University Press.
Percentage of Population with Lactase Persistence
Glycerol kinase
CH2OH HOCH
CH2OH
Mg 2 +
CH2OH HOCH
CH2OPO32–
+
Glycerol sn-Glycerol-3-phosphate
Trang 718.8 How Do Cells Respond to Hypoxic Stress?
Glycolysis is an anaerobic pathway—it does not require oxygen But as noted in
Fig-ure 18.1, operation of the TCA cycle (the subject of Chapter 19) depends on
oxy-gen, so it is aerobic When oxygen is abundant, cells prefer aerobic metabolism,
which yields more energy per glucose consumed However, as Louis Pasteur first
showed, when oxygen is limited, cells adapt to make the most of glycolysis, the less
energetic, anaerobic alternative In mammalian tissues, hypoxia (oxygen limitation)
can cause changes in gene expression that result in increased angiogenesis (the
growth of new blood vessels), increased synthesis of red blood cells, and increased
levels of some glycolytic enzymes (and thus a higher rate of glycolysis)
What is the molecular basis for the increased expression of glycolytic enzymes?
One of the triggers for this expression is a DNA-binding protein called hypoxia
inducible factor (HIF) HIF is a heterodimer of a constitutive nuclear subunit
(HIF-1 ) and an inducible -subunit Both subunits are basic helix-loop-helix
tran-scription factors that bind to hypoxia-inducible genes, and both subunits exist as a
series of isoforms (for example, HIF-1 , HIF-2, and HIF-3) HIF- subunit
regu-lation is a multistep process that includes gene splicing, phosphoryregu-lation,
acetyla-tion, and hydroxylation HIF-1 is the best-studied HIF- isoform When oxygen is
plentiful, HIF-1 is hydroxylated by oxygen-dependent prolyl hydroxylases (PHDs)
at Pro402and Pro564 These hydroxylations ensure its binding to ubiquitin E3 ligase,
which leads to rapid proteolysis by the 26S proteasome (see Chapter 31) HIF-1
binding to the ligase is also promoted by acetylation of Lys532by the ARD1
acetyl-transferase In addition, the presence of oxygen induces the hydroxylation of
HIF-1 Asn803by the hydroxylase factor–inhibiting HIF (FIH-1) Hydroxylation
in-hibits the transcription activity of HIF-1 by preventing its interaction with the
acti-vator p300 Figure 18.27 shows the structure of FIH bound to a fragment of HIF-1 .
Because PHDs and FIH-1 both are oxygen-dependent, lowering oxygen
concen-tration means that HIF-1 avoids degradation and is available to promote gene
tran-scription (Figure 18.28) Phosphorylation of HIF-1 by a protein kinase promotes
Glycerol-P dehydrogenase reaction
H+ +
CH2OH
H OC H
CH2OPO32–
CH2OH C
CH2OPO32–
+
sn-Glycerol-3-phosphate Dihydroxyacetone
phosphate
FIGURE 18.27 FIH (green) bound to HIF.
Oxygen levels
Pro564
HO
Pro564
HO
Degradation
Transcription
Cofactors O2 Fe2+
Pro564
HIF-1
Proteasome
HIF-1
HIF-1
HIF-1
Cofactor PHD
HRE HIF-1
VHL
FIGURE 18.28 The HIF transcription factor is composed
of two subunits: a ubiquitous HIF-1 subunit and a
hypoxia-responsive HIF-1 subunit In response to
hypoxia, inactivation of the PHDs allows HIF-1
stabiliza-tion, dimerization with HIF-1, binding of the dimer to
the hypoxia responsive element (HRE) of HIF target genes, and activation of the transcription of these genes VHL is the von Hippel Lindau subunit of the ubiquitin E3 ligase that targets proteins for proteasome degrada-tion.(Adapted from North, S., Moenner, M., and Bikfalvi, A., 2006 Recent developments in the regulation of the angiogenic
switch by cellular switch factors in tumors Cancer Letters
218:1–14.)
Trang 8binding of HIF-1 to HIF-1, which enhances transcription HIF-1–HIF-1 dimers
bind to hypoxia responsive elements (HREs), activating transcription of
HRE-regulated genes, including genes for glycolytic enzymes Pasteur observed more than 100 years ago that fermentation amounted to “life without air.” The “Pasteur effect” depends on HIF-mediated activation of the genes encoding glycolytic enzymes in the absence of oxygen The linking of glycolytic activity to oxygen level
is the result of an exquisite dance of oxygen-sensitive enzymes with proteins, which undergo covalent modifications that control protein–protein and protein–DNA interactions, a dance that Pasteur could hardly have anticipated.
SUMMARY
Nearly every living cell carries out a catabolic process known as glycolysis—
the stepwise degradation of glucose (and other simple sugars) Glycolysis
is a paradigm of metabolic pathways Localized in the cytosol of cells, it is
basically an anaerobic process; its principal steps occur with no
require-ment for oxygen
18.1 What Are the Essential Features of Glycolysis? Glycolysis
con-sists of two phases In the first phase, a series of five reactions, glucose is
broken down to two molecules of glyceraldehyde-3-phosphate In the
second phase, five subsequent reactions convert these two molecules of
glyceraldehyde-3-phosphate into two molecules of pyruvate Phase 1
consumes two molecules of ATP The later stages of glycolysis result in
the production of four molecules of ATP The net is
4 2 2 molecules of ATP produced per molecule of glucose
18.2 Why Are Coupled Reactions Important in Glycolysis? Coupled
reactions permit the energy of glycolysis to be used for generation of
ATP Conversion of glucose to pyruvate in glycolysis drives the
produc-tion of two molecules of ATP
18.3 What Are the Chemical Principles and Features of the First Phase
of Glycolysis? In the first phase of glycolysis, glucose is converted into
two molecules of glyceraldehyde-3-phosphate First, glucose is
phos-phorylated to glucose-6-P, which is isomerized to fructose-6-P Another
phosphorylation and then cleavage yields two 3-carbon intermediates
One of these is glyceraldehyde-3-P, and the other, dihydroxyacetone-P,
is converted to glyceraldehyde-3-P Energy released from this
high-energy molecule in the second phase of glycolysis is then used to
syn-thesize ATP
18.4 What Are the Chemical Principles and Features of the Second Phase
of Glycolysis? The second half of the glycolytic pathway involves the
re-actions that convert the metabolic energy in the glucose molecule into
ATP Phase 2 starts with the oxidation of glyceraldehyde-3-phosphate, a
re-action with a large enough energy “kick” to produce a high-energy
phos-phate, namely, 1,3-bisphosphoglycerate Phosphoryl transfer from 1,3-BPG
to ADP to make ATP is highly favorable The product, 3-phosphoglycerate,
is converted via several steps to phosphoenolpyruvate (PEP), another
high-energy phosphate PEP readily transfers its phosphoryl group to ADP
in the pyruvate kinase reaction to make another ATP
18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in
Glycolysis? In addition to ATP, the products of glycolysis are NADH
and pyruvate Their processing depends upon other cellular pathways
NADH must be recycled to NAD, lest NADbecome limiting in
gly-colysis NADH can be recycled by both aerobic and anaerobic paths,
ei-ther of which results in furei-ther metabolism of pyruvate What a given cell does with the pyruvate produced in glycolysis depends in part on the availability of oxygen Under aerobic conditions, pyruvate can be sent into the citric acid cycle, where it is oxidized to CO2with the pro-duction of additional NADH (and FADH2) Under aerobic conditions, the NADH produced in glycolysis and the citric acid cycle is reoxidized
to NADin the mitochondrial electron-transport chain
Under anaerobic conditions, the pyruvate produced in glycolysis is not sent to the citric acid cycle Instead, it is reduced to ethanol in yeast;
in other microorganisms and in animals, it is reduced to lactate These processes are examples of fermentation—the production of ATP energy
by reaction pathways in which organic molecules function as donors and acceptors of electrons In either case, reduction of pyruvate provides
a means of reoxidizing the NADH produced in the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis
18.6 How Do Cells Regulate Glycolysis? The standard-state free en-ergy changes for the 10 reactions of glycolysis are variously positive and negative and, taken together, offer little insight into the coupling that occurs in the cellular milieu On the other hand, the values of G
un-der cellular conditions fall into two distinct classes For reactions 2 and
4 through 9, G is very close to zero, meaning these reactions operate
essentially at equilibrium Small changes in the concentrations of reac-tants and products could “push” any of these reactions either forward
or backward By contrast, the hexokinase, phosphofructokinase, and pyruvate kinase reactions all exhibit large negative G values under
cel-lular conditions These reactions are thus the sites of glycolytic regula-tion
18.7 Are Substrates Other Than Glucose Used in Glycolysis? Fructose enters glycolysis by either of two routes Mannose, galactose, and glyc-erol enter via reactions that are linked to the glycolytic pathway
18.8 How Do Cells Respond to Hypoxic Stress? Glycolysis is an anaer-obic pathway, but it normally feeds pyruvate into aeranaer-obic metabolic pathways However, when oxygen is limited, cells adapt to make the most of glycolysis In mammalian tissues, oxygen limitation (hypoxia) can cause changes in gene expression that result in increased angio-genesis, red blood cell synthesis, and elevated levels of some glycolytic enzymes One of the triggers for this expression is a DNA-binding pro-tein, HIF, which binds to hypoxia-inducible genes HIF- regulation is a
multistep process that includes gene splicing, phosphorylation, acetyla-tion, and hydroxylation The Pasteur effect depends on HIF-mediated activation of the genes encoding glycolytic enzymes in the absence of oxygen
Trang 9Preparing for an exam? Create your own study path for this
chapter at www.cengage.com/login
1.List the reactions of glycolysis that
a are energy consuming (under standard-state conditions)
b are energy yielding (under standard-state conditions)
c consume ATP
d yield ATP
e are strongly influenced by changes in concentration of substrate
and product because of their molecularity
f are at or near equilibrium in the erythrocyte (see Table 18.2)
2.Determine the anticipated location in pyruvate of labeled carbons
if glucose molecules labeled (in separate experiments) with 14C at
each position of the carbon skeleton proceed through the glycolytic
pathway
3.In an erythrocyte undergoing glycolysis, what would be the effect of
a sudden increase in the concentration of
c fructose-1,6-bisphosphate? d fructose-2,6-bisphosphate?
e citrate? f glucose-6-phosphate?
4.Discuss the cycling of NADH and NADin glycolysis and the related
fermentation reactions
5.For each of the following reactions, name the enzyme that carries
out this reaction in glycolysis and write a suitable mechanism for the
reaction
6.Write the reactions that permit galactose to be utilized in glycolysis
Write a suitable mechanism for one of these reactions
7.(Integrates with Chapters 4 and 14.) How might iodoacetic acid
affect the glyceraldehyde-3-phosphate dehydrogenase reaction in
glycolysis? Justify your answer
8.If32P-labeled inorganic phosphate were introduced to erythrocytes
undergoing glycolysis, would you expect to detect 32P in glycolytic
intermediates? If so, describe the relevant reactions and the 32P
incorporation you would observe
9.Sucrose can enter glycolysis by either of two routes:
Sucrose phosphorylase:
Sucrose Pi34 fructose glucose-1-phosphate
Invertase:
Sucrose H2O34 fructose glucose
Would either of these reactions offer an advantage over the other in
the preparation of hexoses for entry into glycolysis?
10.What would be the consequences of a Mg2ion deficiency for the
reactions of glycolysis?
HOCH
HCOH
HCOH
CH2OPO3
CH2OPO3
CH2OPO3
CH2OH
CHO HCOH
CH2OPO3
CHO HCOH
CH2OPO3
C HCOH
CH2OPO3
OPO3 O
11. (Integrates with Chapter 3.) Triose phosphate isomerase catalyzes the conversion of dihydroxyacetone-P to glyceraldehyde-3-P The standard free energy change, G°, for this reaction is 7.6 kJ/mol.
However, the observed free energy change (G) for this reaction in
erythrocytes is 2.4 kJ/mol
a Calculate the ratio of [dihydroxyacetone-P]/[glyceraldehyde-3-P]
in erythrocytes from G.
b If [dihydroxyacetone-P] 0.2 mM, what is [glyceraldehyde-3-P]?
12. (Integrates with Chapter 3.) Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate H2O The standard free energy change, G°, for this reaction is 1.8 kJ/mol If the concentration of 2-phosphoglycerate is 0.045 mM and the concen-tration of phosphoenolpyruvate is 0.034 mM, what is G, the free
energy change for the enolase reaction, under these conditions?
13. (Integrates with Chapter 3.) The standard free energy change (G°) for hydrolysis of phosphoenolpyruvate (PEP) is 61.9 kJ/mol.
The standard free energy change (G°) for ATP hydrolysis is
30.5 kJ/mol
a What is the standard free energy change for the pyruvate kinase reaction:
ADP phosphoenolpyruvate⎯⎯→ATP pyruvate
b What is the equilibrium constant for this reaction?
c Assuming the intracellular concentrations of [ATP] and [ADP]
remain fixed at 8 mM and 1 mM, respectively, what will be the
ratio of [pyruvate]/[phosphoenolpyruvate] when the pyruvate ki-nase reaction reaches equilibrium?
14. (Integrates with Chapter 3.) The standard free energy change (G°) for hydrolysis of 1,6-bisphosphate (FBP) to
fructose-6-phosphate (F-6-P) and Piis16.7 kJ/mol:
FBP H2O⎯⎯→fructose-6-P Pi
The standard free energy change (G°) for ATP hydrolysis is
30.5 kJ/mol:
ATP H2O⎯⎯→ADP Pi
a What is the standard free energy change for the phosphofructo-kinase reaction:
ATP fructose-6-P⎯⎯→ADP FBP
b What is the equilibrium constant for this reaction?
c Assuming the intracellular concentrations of [ATP] and [ADP]
are maintained constant at 4 mM and 1.6 mM, respectively, in a rat
liver cell, what will be the ratio of [FBP]/[fructose-6-P] when the phosphofructokinase reaction reaches equilibrium?
15. (Integrates with Chapter 3.) The standard free energy change (G°) for hydrolysis of 1,bisphosphoglycerate (1,BPG) to
3-phosphoglycerate (3-PG) and Piis49.6 kJ/mol:
1,3-BPG H2O⎯⎯→3-PG Pi
The standard free energy change (G°) for ATP hydrolysis is
30.5 kJ/mol:
ATP H2O⎯⎯→ADP Pi
a What is the standard free energy change for the phosphoglycerate kinase reaction:
ADP 1,3-BPG⎯⎯→ATP 3-PG
b What is the equilibrium constant for this reaction?
c If the steady-state concentrations of [1,3-BPG] and [3-PG] in an erythrocyte are 1
ratio of [ATP]/[ADP], assuming the phosphoglycerate kinase re-action is at equilibrium?
Trang 1016. The standard-state free energy change, G°, for the hexokinase
re-action is 16.7 kJ/mol Use the values in Table 18.2 to calculate the
value of G for this reaction in the erythrocyte at 37°C.
17. Taking into consideration the equilibrium constant for the
adeny-late kinase reaction (page 542), calcuadeny-late the change in
concentra-tion in AMP that would occur if 8% of the ATP in an erythrocyte
(red blood cell) were suddenly hydrolyzed to ADP In addition to
the concentration values in Table 18.2, it may be useful to assume
that the initial concentration of AMP in erythrocytes is 5
18. Fructose bisphosphate aldolase in animal muscle is a class I aldolase,
which forms a Schiff base intermediate between substrate (for
ex-ample, fructose-1,6-bisphosphate or dihydroxyacetone phosphate)
and a lysine at the active site (see Figure 18.12) The chemical
evi-dence for this intermediate comes from studies with aldolase and
the reducing agent sodium borohydride, NaBH4 Incubation of the
enzyme with dihydroxyacetone phosphate and NaBH4 inactivates
the enzyme Interestingly, no inactivation is observed if NaBH4is
added to the enzyme in the absence of substrate Write a mechanism
that explains these observations and provides evidence for the
for-mation of a Schiff base intermediate in the aldolase reaction
19. As noted on page 556, the galactose-1-phosphate uridylyltransferase
reaction proceeds via a ping-pong mechanism Consult Chapter 13,
page 406, to refresh your knowledge of ping-pong mechanisms, and
draw a diagram to show how a ping-pong mechanism would pro-ceed for the uridylyltransferase
20.Genetic defects in glycolytic enzymes can have serious conse-quences for humans For example, defects in the gene for pyruvate kinase can result in a condition known as hemolytic anemia Con-sult a reference to learn about hemolytic anemia, and discuss why such genetic defects lead to this condition
Preparing for the MCAT Exam
21.Regarding phosphofructokinase, which of the following statements
is true:
a Low ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits
b High ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits
c High ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits
d The enzyme is more active at low ATP than at high, and fructose-2,6-bisphosphate activates the enzyme
e ATP and fructose-2,6-bisphosphate both inhibit the enzyme
22.Based on your reading of this chapter, what would you expect to be the most immediate effect on glycolysis if the steady-state
concen-tration of glucose-6-P were 8.3 mM instead of 0.083 mM ?
FURTHER READING
General
Fothergill-Gilmore, L., 1986 The evolution of the glycolytic pathway
Trends in Biochemical Sciences 11:47–51.
Kim, J-W., and Dang, C V., 2006 Cancer’s molecular sweet tooth and
the Warburg effect Cancer Research 66:8927–8930.
Sparks, S., 1997 The purpose of glycolysis Science 277:459–460.
Waddell, T G., 1997 Optimization of glycolysis: A new look at the
effi-ciency of energy coupling Biochemical Education 25:204–205.
Enzymes of Glycolysis
Aleshin, A E., Kirby, C., et al., 2000 Crystal structures of mutant
monomeric hexokinase I reveal multiple ADP-binding sites and
conformational changes relevant to allosteric regulation Journal of
Molecular Biology 296:1001–1015.
Choi, K H., Shi, J., et al., 2001 Snapshots of catalysis: The structure of
fructose-1,6-(bis)phosphate aldolase covalently bound to the
sub-strate dihydroxyacetone phosphate Biochemistry 40:13868–13875.
Didierjean, C., Corbier, C., et al., 2003 Crystal structure of two ternary
complexes of phosphorylating glyceraldehyde-3-phosphate
dehydro-genase from Bacillus sterothermophilus with NAD and D-glyceraldehyde
3-phosphate Journal of Biological Chemistry 278:12968–12976.
Jeffery, C J., 1999 Moonlighting proteins Trends in Biochemical Sciences
24:8–11
Jeffery, C J., 2004 Molecular mechanisms for multitasking: Recent
crys-tal structures of moonlighting proteins Current Opinion in Structural
Biology 14:663–668.
Kim, J-W., and Dang, C V, 2005 Multifaceted roles of glycolytic
en-zymes Trends in Biochemical Sciences 30:142–150.
Lee, J H., Chang, K Z., et al., 2001 Crystal structure of rabbit
phos-phoglucose isomerase complexed with its substrate D-fructose
6-phosphate Biochemistry 40:7799–7805.
Lolis, E., and Petsko, G., 1990 Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5 Å
resolution: Implications for catalysis Biochemistry 29:6619–6625.
Schirmer, T., and Evans, P R., 1999 Structural basis of the allosteric
be-haviour of phosphofructokinase Nature 343:140–145.
Valentini, G., Chiarelli, L., et al., 2000 The allosteric regulation of
pyru-vate kinase Journal of Biological Chemistry 275:18145–18152.
Wilson, J E., 2003 Isozymes of mammalian hexokinase: Structure,
sub-cellular localization and metabolic function Journal of Experimental
Biology 206:2049–2057.
Zhang, E., Brewer, J M., et al., 1997 Mechanism of enolase: The crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/
enolase-phosphoenopyruvate at 2.0 Å resolution Biochemistry 36:
12526–12534
Muscle Biochemistry
Green, H J., 1997 Mechanisms of muscle fatigue in intense exercise
Journal of Sports Sciences 15:247–256.
HIF-1␣ and Glycolysis
Cramer, T., Yamanishi, Y., et al., 2003 HIF-1 is essential for myeloid
cell-mediated inflammation Cell 112:645–657.
Melillo, G., 2006 Inhibiting hypoxia-inducible factor 1 for cancer
ther-apy Molecular Cancer Research 4:601–605.
Melillo, G., 2007 Targeting hypoxia cell signaling for cancer therapy
Cancer and Metastasis Reviews 26:341–352.
North, S., Moenner, M., et al., 2005 Recent developments in the regu-lation of the angiogenic switch by cellular stress factors in tumors
Cancer Letters 218:1–14.