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UNIT II:
Intermediary Metabolism
Glycolysis
Overview
• In cells reactions rarely occur in isolation, but rather
organized into multi-step sequences called pathways, such
as glycolysis
• In a pathway, product of one reaction serves as substrate of
subsequent reaction
• Different pathways can also intersect, forming an integrated
and purposeful network of chemical reactions. These are
collectively called metabolism, which is the sum of all
chemical changes occurring in a cell, a tissue or the body.
• Most pathways can be classified as either catabolic
(degradative) or anabolic (synthetic).
• Catabolic reactions break down complex molecules e.g.,
proteins, polysacch’s & lipids, to a few simple molecules
e.g., CO2, NH3 & H2O
• Anabolic pathway form complex end products from simple
precursors e.g., synthesis of the polysacch, glycogen, from
glucose
Figure 8.1
Glycolysis, an example of a
metabolic pathway.
A. Metabolic map
- It is convenient to investigate metabolism by
-
-
examining its component pathways
Each pathway is multienzyme sequences, and
each enz in turn may exhibit important catalytic
or regulatory feature
Metabolic map is useful in tracing connections
b/w pathways, visualizing purposeful movement
of metabolic intermediates, and picturing the
effect on flow of intermediates if a pathway is
blocked e.g., by a drug or inherited deficiency of
an enz
Figure 8.2. Important reactions
of intermediary metabolism.
Several important pathways to
be discussed in later chapters
are highlighted. Curved
reaction arrows (
)
indicate forward and reverse
reactions that are catalyzed by
different enzymes.
The straight arrows (
)
indicate forward and reverse
reactions that are
catalyzed by the same
enzyme. Key: Blue text =
intermediates of carbohydrate
metabolism; brown text =
intermediates of lipid
metabolism; green text =
intermediates of protein
metabolism.
B. Catabolic pathways
- Catabolic reactions serve to capture chemical energy in
the form of ATP from degradation of energy-rich fuel
molecules
- Catabolism also allows molecules in diet (or nutrient
molecules stored in cells) to be converted to building
blocks needed for synthesis of complex molecules
- Energy generation by degradation of complex molecules
occurs in 3 stages:
1. Hydrolysis of complex molecules: complex molecules
are broken down into their component building blocks.
E.g., proteins  aa’s, polysacch’s  monosacch’s &
triglycerides  free fatty acids and glycerol
2. Conversion of building blocks to simple intermediates:
diverse building blocks further degraded to acetyl CoA
and a few other simple molecules.
Some energy is captured as ATP, but amount is small
compared with that produced during 3rd stage
3. Oxidation of acetyl CoA: TCA cycle is the final common
pathway in oxidation of fuel molecules such as acetyl
CoA.
Large amounts of ATP are generated as e’s flow from
NADH & FADH2 to O2 via oxphos
Figure 8.3. Three stages of catabolism.
C. Anabolic pathway
- Anabolic reactions combine small molecules, e.g., aa’s
to form complex molecules, such as proteins
- Anabolic reactions require energy, which is generally
provided by breakdown of ATP to ADP + Pi
- Anabolic reactions often involve chemical reductions in
which reducing power is most frequently provided by edonor NADPH
- Note that catabolism is a convergent process i.e., a wide
variety of molecules are transformed into a few common
end products
- By contrast, anabolism is a divergent process i.e., a few
biosynthetic precursors form a wide variety of polymeric
or complex products
Figure 8.4. Comparison
of catabolic and
anabolic pathways.
II. Regulation of metabolism
- Pathways of metabolism must be coordinated so
that production of energy or synthesis of end
products meets needs of cell
- Individual cells do not function in isolation but,
rather, are part of a community of interacting
tissues
- Thus, a sophisticated communication system
has evolved to coordinate functions of the body
- Regulatory signals that inform an individual cell
of the metabolic state of the body as a whole
include hormones, neurotransmitters, and the
availability of nutrients. These, in turn, influence
signals generated within the cell
A. Signals from within the cell (intracellular)
- Rate of a metabolic pathway can respond to
regulatory signals that arise from within cell
- E.g., rate of a pathway may be influenced by
availability of substrates, product inhibition, or
alterations in levels of allosteric activators or
inhibitors
- These intracellular signals typically elicit rapid
responses, and are important for moment-tomoment regulation of metabolism
B. Communication between cells (intercellular)
Ability to respond to extracellular signals is essential
for survival & development of all organisms
Signaling b/w cells provides for long-range integration
of metabolism, & usually results in a response that is
slower than is seen with signals that originates within
the cell
Communication b/w cells can be mediated by surfaceto-surface contact &, in some tissues, by formation of
gap junctions, allowing direct communication b/w
cytoplasms of adjacent cells
However, for energy metabolism, the most important
route of communication is chemical signaling e.g., by
blood-borne hormones or by neurotransmitters
C. Second messenger systems
- Hormones or neurotransmitters can be thought of as
signals, & a receptor as a signal detector. Each
component serves as a link in the communication b/w
extracellular events & chemical changes within the cell
- Many receptors signal their recognition of a bound ligand
by initiating a series of reactions that ultimately result in
a specific intracellular response
- “second messenger” molecules, so named as they
intervene b/w original messenger (neurotransmitter or
hormone) & the ultimate effect on cell, are part of the
cascade of events that translates hormone or
neurotransmitter binding into a cellular response
- Two of the most widely recognized 2nd messenger
systems are the calcium/phosphatidylinositol system &
adenylyl cyclase system, which is particularly important
in regulating pathways of intermediary metabolism
Figure 8.5
Some commonly used
mechanisms for transmission
of regulatory signals between
cells.
D. Adenylyl cyclase
- Recognition of a chemical signal by some memb
receptors, such as β- & α2-adrenergic receptors, triggers
either an increase or a decrease in the activity of
adenylyl cyclase.
- This is a memb-bound enz that converts ATP to 3`,5`adenosine monophosphate (a.k.a cyclic AMP or cAMP)
- Chemical signals are most often hormones or
neurotransmitters, each of which binds to a unique type
of memb receptor
- Therefore, tissues that respond to more than one
chemical signal must have several different receptors,
each of which can be linked to adenylyl cyclase
Note: certain toxins, as that produced by Vibrio cholera,
can also activate the adenylyl cyclase cascade, with
potentially disastrous consequences
- These receptors are characterized by an extracellular
ligand-binding region, 7 transmembrane helices, & an
intracellular domain that interacts with G-proteins
Figure 8.6
Structure of a
typical membrane
receptor.
1.
-
-
-
GTP-dependent regulatory proteins:
Effect of activated occupied receptor on 2nd messenger
formation is not direct, it is mediated by specialized
trimeric proteins in the CM.
These, referred to as G-proteins because they bind
guanosine nucleotides (GTP & GDP), form a link in the
chain of communication b/w receptor & adenylyl
cyclase
The inactive form of G-protein binds to GDP
The activated receptor interacts with G-proteins,
triggering an exchange of GTP for GDP.
The timeric G-proteins then dissociates into an α
subunit & a βγ dimer.
The GTP-bound form of the α subunit moves from the
receptor to adenylyl cyclase, which is thereby activated
- Many molecules of active G-protein are formed by one
activated receptor
Note: ability of a hormone or neurotransmitter to stimulate
or inhibit adenylyl cyclase depends on the type of Gprotein that is linked to the receptor. One family of Gproteins, designated Gs, is specific for stimulation of
adenylyl cyclase; another family, Gi  inhibition of the
enz.
- Actions of G-protein-GTP complex are short-lived
because the G-protein has an inherent GTPase activity
 rapid hydrolysis of GTP to GDP  inactivation of Gprotein
Figure 8.7. The recognition of chemical signals by certain
membrane receptors triggers an increase (or, less often, a
decrease) in the activity of adenylyl cyclase.
2. Protein kinases:
- Next link in cAMP 2nd-messenger system is activation by
cAMP of a family of enz’s = cAMP-dependent protein
kinases, e.g., protein kinase A.
- cAMP activates protein kinase A by binding to its two
regulatory subunits, causing release of active catalytic
subunits.
- The active subunits catalyze the transfer of P from ATP
to specific ser or thr residues of protein substrates
- Phosphorylated proteins may act directly on cell’s ion
channels, or may become activated or inhibited enz’s
- Protein kinase A can also phosphorylate specific proteins
that bind to promoter regions of DNA  increased
expression of specific genes
- Note: not all protein kinases respond to cAMP; there are
several types of protein kinases that are not cAMPdependent, e.g., protein kinase C
Figure 8.8. Actions of cAMP.
3. Dephosphorylation of proteins:
- P groups added to proteins by protein kinases are
removed by protein phosphatases, enz’s that
hydrolytically cleave phosphate esters
- This ensures that changes in enzymatic activity induced
by protein phosphorylation are not permanent
4. Hydrolysis of cAMP:
- cAMP is rapidly hydrolyzed to 5`-AMP by cAMP
phosphodiesterase, one of a family of enz’s that cleave
cyclic 3`,5`-phosphodiester bond
- 5`-AMP is not an intracellular signaling molecule. Thus,
effects of neurotransmitter or hormone-mediated
increases of cAMP are rapidly terminated if the
extracellular signal is removed
Note: phosphodiesterase is inhibited by methylxanthine
derivatives, such as theophylline & caffeine
III. Overview of glycolysis
- Glycolytic pathway is employed by all tissues for the
breakdown of glucose to provide energy (in form of ATP)
and intermediates for other metabolic pathways
- Glycolysis is at the hub of CHO metabolism because
virtually all sugars, whether arising from diet or from
catabolic reactions in the body, can ultimately be
converted to glucose
- Pyruvate is the end product of glycolysis in cells with
mitochondria & an adequate supply of oxygen
- This series of 10 reactions is called aerobic glycolysis
because oxygen is required to reoxidize NADH formed
during oxidation of glyceraldehyde-3-P
- Aerobic glycolysis sets the stage for the oxidative
decarboxylation of pyruvate to acetyl CoA, a major fuel
of TCA cycle
- Alternatively, glucose can be converted to pyruvate,
which is reduced by NADH  lactate. This is called
anaerobic glycolysis because it can occur without
participation of oxygen.
- Anaerobic glycolysis allows the continued production of
ATP in tissues that lack mitochondria (e.g., RBCs) or in
cells deprived of sufficient oxygen
Figure 8.9. A. Glycolysis shown as one of the essential pathways of
energy metabolism. B. Reactions of aerobic glycolysis. C. Reactions of
anaerobic glycolysis.
IV. Transport of glucose into cells
-
Glucose cannot diffuse directly into cells, but enters by
one of two transport mechanisms: a Na+-independent,
facilitated diffusion transport system, or a Na+monosacch co-transporter system
A. Na+-independent facilitated diffusion transport
This system is mediated by a family of at least 14
glucose transporters in CMs. They are = GLUT-1 to
GLUT-14 (glucose transporter isoform 1-14)
These transporters exist in membrane in two
conformational states. Extracellular glucose binds to
the transporter, which then alters its conformation,
transporting glucose across the CM
Figure 8.10
Schematic
representation of the
facilitated transport
of glucose through a
cell membrane.
1. Tissue specificity of GLUT gene expression:
- Glucose transporters display a tissue-specific pattern of
expression e.g., GLUT-3 is the primary glucose
transporter in neurons. GLUT-1 is abundant in
erythrocytes & brain, but is low in adult muscle,
whereas GLUT-4 is abundant in adipose tissue &
skeletal muscle
Note: number of GLUT-4 transporters active in these
tissues is increased by insulin. The other GLUT
isoforms also have tissue-specific distributions
2. Specialized functions of GLUT isoforms:
- In facilitated diffusion, glucose movement follows a conc.
gradient, i.e., from a high gluc conc. to a lower one. E.g.,
GLUT-1, -3, -4 are primarily involved in gluc uptake from
blood
- In contrast, GLUT-2 which is found in liver, kidney, and βcells of pancreas, can either transport gluc into these
cells when blood gluc levels are high, or transport gluc
from cells to blood when blood glucose levels are low
(e.g., during fasting)
- GLUT-5 is unusual in that it is the primary transporter for
fructose (instead of gluc) in the small intestine & testes
- GLUT-7 which is expressed in liver & other
gluconeogenic tissues, mediates glucose flux across
endoplasmic reticulum memb.
B. Na+-monosaccharide cotransporter system
- This is an energy-requiring process that
transports glucose “against” a conc. gradient i.e.,
from low gluc conc’s outside the cell to higher
conc’s within cell
- This system is a carrier-mediated process in
which movement of glucose is coupled to the
conc gradient of Na+, which is transported into
cell at the same time.
- This type of transport occurs in epithelial cells of
intestine, renal tubules, and choroid plexus
V. Reactions of glycolysis
• Conversion of gluc to pyruvate occurs in 2 stages. The 1st
five reactions correspond to an energy investment phase
in which phosphorylated forms of intermediates are
synthesized at the expense of ATP
• The subsequent reactions of glycolysis constitute an
energy generation phase in which a net of 2 molecules of
ATP are formed by substrate level phosphorylation per
gluc molecule metabolized
Note: 2 molecules of NADH are formed when pyruvate is
produced (aerobic glycolysis), whereas NADH is
converted to NAD+ when lactate is produced (anaerobic
glycolysis)
Figure 8.11
Two phases of aerobic
glycolysis.
A. Phosphorylation of glucose
- Phosphorylated sugar molecules do not readily
penetrate CMs, because no specific
transmemb carriers for these cpds, & they are
too polar to diffuse through the CM
- The irreversible phosphorylation of gluc,
therefore, effectively traps the sugar as
cytosolic G-6-P, thus committing it to further
metabolism in the cell
- Mammals have several isozymes of the enz
hexokinase that catalyze the phosphrylation of
gluc to G-6-P
Figure 8.12
Energy investment
phase: phosphorylation of
glucose.
1. Hexokinase:
In most tissues, phosphorylation of gluc is catalyzed by
hexokinase, one of 3 regulatory enz’s of glycolysis
(phosphofructokinase I & pyruvate kinase are the other
two)
Hexokinase has broad substrate specificity and is able
to phosphorylate several hexoses in addition to gluc.
Hexokinase is inhibited by the reaction product, G-6-P,
which accumulates when further metabolism of this
hexose-P is reduced
Hexokinase has a low Km (&, therefore, a high affinity)
for gluc. This permits efficient phosphorylation &
subsequent metabolism of gluc even when tissue
conc’s of gluc are low
Hexokinase, however, has a low Vmax for gluc &,
therefore, cannot sequester (trap) cellular phosphate in
the form of phosphorylated hexoses, or phosphorylate
more sugars than the cell can use
2. Glucokinase:
- In liver parenchymal cells & islet cells of the
pancreas, glucokinase (also called hexokinase
D, or type IV) is the predominant enz
responsible for the phosphorylation of gluc
- In β-cells, glucokinase functions as gluc sensor,
determining the threshold for insulin secretion
- In liver, the enz facilitates gluc phosphorylation
during hyperglycemia
Note: despite its misleading name “glucokinase”
the sugar specificity of the enz is similar to that
of other hexokinase isozymes
a. Kinetics:
Glucokinase differs from hexokinase in several
important properties
-
-
E.g., it has much higher Km, requiring a higher gluc conc for
half-saturation. Thus glucokinase functions only when
intracellular conc of gluc in hepatocyte is elevated, e.g., during
the brief period following consumption of a CHO-rich meal,
when high levels of gluc are delivered to the liver via the portal
vein
Glucokinase has a high Vmax, allowing the liver to effectively
remove the flood of gluc delivered by the portal blood. This
prevents large amounts of gluc from entering systemic
circulation following a CHO-rich meal, & thus minimizes
hyperglycemia during the absorptive period
Note: GLUT-2 insures that blood gluc equilibrates rapidly
across the memb of the hepatocyte
Figure 8.13
Effect of glucose
concentration on the
rate of phosphorylation
catalyzed by
hexokinase and
glucokinase.
b. Regulation by fructose 6-phosphate and glucose:
- Glucokinase activity is not allosterically inhibited by G-6-P as
are other hexokinases, but rather is indirectly inhibited by F-6P (which is in equil. with G-6-P), & is stimulated indirectly by
glucose via the following mechanism:
- A glucokinase regulatory protein exists in the nucleus of
hepatocytes.
- In presence of F-6-P, glucokinase is translocated into nucleus &
binds tightly to regulatory protein, thus rendering enz inactive
- When gluc levels in the blood (& also in the hepatocyte, as a
result of GLUT-2) increase, the gluc causes the release of
glucokinase from the regulatory protein, & the enz enters the
cytosol where it phosphorylates gluc to G-6-P
- As free gluc levels fall, F-6-P causes glucokinase to translocate
back into nucleus & bind to regulatory protein, thus inhibiting
enz’s activity
Figure 8.14
Regulation of
glucokinase activity by
glucokinase regulatory
protein.
c. Regulation by insulin:
- Glucokinase activity in hepatocytes is also increased by insulin
- as blood gluc levels rise following a meal, β-cells of
pancreas are stimulated to release insulin into portal
circulation
Note: ~ ½ of the newly secreted insulin is extracted by liver
during the 1st pass through that organ. Therefore, liver is
exposed to twice as much insulin as is found in systemic
circulation
- insulin also promotes transcription of glucokinase gene,
resulting in an increase in liver enz protein &, therefore, of
total glucokinase activity.
Note: the absence of insulin in patients with diabetes causes a
deficiency in hepatic glucokinase. This contributes to an
inability of patient to efficiently decrease blood glucose levels
B. Isomerization of glucose-6-phosphate
- Isomerization of G-6-P to F-6-P is catalyzed by
phosphoglucose isomerase. Reaction is readily
reversible & is not a rate-limiting or regulated step
Figure 8.15
Isomerization of
glucose 6-phosphate to
fructose 6-phosphate.
C. Phosphorylation of fructose 6-phosphate
- The irreversible phosphorylation reaction catalyzed
by phosphofructokinase-1 (PFK-1) is the most
important control point & the rate-limiting step of
glycolysis
- PFK-1 is controlled by available conc’s of the
substrates ATP & F-6-P, & by the following
regulatory substances:
1. Regulation by energy levels within the cell:
- PFK-1 is inhibited allosterically by elevated levels of
ATP, which act as an “energy-rich” signal indicating an
abundance of high-energy cpds.
- Elevated levels of citrate, an intermediate in the TCA
cycle, also inhibit PFK-1. Conversely, PFK-1 is
activated allosterically by high conc’s of AMP, which
signal that the cells’ energy stores are depleted
2. Regulation by fructose 2,6-bisphosphate
- F-2,6-bisP is the most potent activator of PFK-1.
This cpd also acts as an inhibitor of fructose 1,6bisphosphatase
- The reciprocal actions of F-2,6-bisP on glycolysis &
gluconeogenesis ensure that both pathways are not
fully active at the same time
Note: this would result in a “futile cycle” in which
glucose would be converted to pyruvate followed by
re-synthesis of glucose from pyruvate
- F-2,6-bisP is formed PFK-2, an enz different than
PFK-1. F-2,6-bisP is converted back to F-6-P by
fructose bisphosphatase-2
Note: the kinase & phosphatase activities are different
domains of one bifunctional polyp molecule
Figure 8.16
Energy investment phase
(continued): Conversion of
fructose 6-phosphate to
triose phosphates.
a. During the well-fed state:
- Decreased levels of glucagon & elevated
levels of insulin, such as occur following a
CHO-rich meal, cause an increase in F-2,6bisP and thus the rate of glycolysis in the liver.
- F-2,6-bisP, therefore acts as an intracellular
signal, indicating that glucose is abundant
b. During starvation:
- Elevated levels of glucagon & low levels of
insulin, such as occur during fasting, decrease
the intracellular conc of hepatic F-2,6-bisP.
This results in a decrease in the overall rate of
glycolysis and an increase in gluconeogenesis
Figure 8.17. Effect of elevated insulin concentration on the
intracellular concentration of fructose 2,6-bisphosphate in liver.
PFK-2 = phosphofructokinase-2; FBP-2 = Fructose bisphospate
phosphatase-2.
D. Cleavage of fructose 1,6-bisphosphate
- Aldolase A cleaves F-1,6-bisP to dihydroxyacetoneP (DHAP) & glyceraldehyde-3-P (GA-3P). The
reaction is reversible & not regulated
Note: Aldolase B in the liver and kidney also
cleaves F-1,6-bisP, & functions in metabolism of
dietary fructose
E. Isomerization of dihydroxyacetone phosphate
- Triose phosphate isomerase interconverts DHAP
& GA-3P. DHAP must be isomerized to GA-3P for
further metabolism by glycolytic pathway
- This isomerization results in net production of 2
GA-3P from cleavage products of F-1,6-bisP
Figure 8.16
Energy investment phase
(continued): Conversion of
fructose 6-phosphate to
triose phosphates.
F. Oxidation of glyceraldehyde 3-phosphate
- Conversion of GA-3P to 1,3-bisP glycerate by
GA-3P dehydrogenase is 1st redox reaction of
glycolysis
Note: because there is only a limited amount of
NAD+ in the cell, NADH formed by this reaction
must be reoxidized to NAD+ for glycolysis to
continue.
- Two major mechanisms for oxidizing NADH are:
1) NADH-linked conversion of pyruvate to lactate
2) oxidation of NADH via respiratory chain
1. Synthesis of 1,3-bisphosphoglycerate (1,3-BPG):
- Oxidation of aldehyde group of GA-3P to a
carboxyl group is coupled to attachment of Pi to
the carboxyl group
- The high-energy P group at C-1 of 1,3-BPG
conserves much of the free energy produced by
oxidation of GA-3P
- The energy of this high-energy P drives
synthesis of ATP in the next reaction of
glycolysis
2. Mechanism of arsenic poisoning:
- Toxicity of arsenic is explained primarily by inhibition
of enz’s such as pyruvate dehydrogenase, which
require lipoic acid as a cofactor. However,
pentevalent arsenic (arsenate) also prevents net
ATP & NADH production by glycolysis, without
inhibiting the pathway itself.
- The poison does so by competing with Pi as a
substrate for GA-3P dehydrogenase, forming a
complex that spontaneously hydrolyzes to form 3-Pglycerate
- By bypassing synthesis and dephosphorylation of
1,3-BPG, the cell is deprived of energy usually
obtained from glycolytic pathway
3. Synthesis of 2,3-BPG in RBCs:
- Some 1,3-BPG is converted to 2,3-BPG by bisphosphoglycerate mutase
- 2,3-BPG, which is found in only trace amounts in most
cells, is present at high conc in RBCs
- 2,3 BFG is hydrolyzed by a phosphatase to 3phosphoglycerate, which is also an intermediate in
glycolysis
- In RBCs, glycolysis is modified by inclusion of these
“shunt” reactions
Figure 8.18. Energy-generating phase: conversion of
glyceraldehyde 3-phosphate to pyruvate.
G. Synthesis of 3-phosphoglycerate producing ATP
- When 1,3-BPG is converted to 3-phosphoglycerate,
the high-energy P group of 1,3-BPG is used to
synthesize ATP from ADP
- This reaction is catalyzed by phosphoglycerate
kinase, which, unlike most other kinases, is
physiologically reversible
- Because 2 molecules of 1,3-BPG are formed from
each gluc molecule, this kinase reaction replaces
the 2 ATP molecules consumed by earlier formation
of G-6-P & F-1,6-BP
Note: this is an example of substrate level phospho., in
which production of a high-energy P is coupled
directly to oxidation of substrate, instead of resulting
from oxidative phosph. via ETC.
H. Shift of phosphate group from carbon 3 to carbon 2
Shift of P group from C-2 to C-3 of
phosphoglycerate by phosphoglycerte mutase is
freely reversible
I. Dehydration of 2-phosphoglycerate
Dehydration of 2-phosphoglycerate by enolase
distributes the energy within 2-phosphoglycerate
 formation of PEP, which contains a highenergy enol phosphate. The reaction is
reversible despite the high-energy nature of the
product
J. Formation of pyruvate producing ATP
Conversion of PEP to pyruvate is catalyzed by
pyruvate kinase. The 3rd irreversible reaction of
glycolysis.
The equil of pyruvate kinase reaction favors formation
of ATP
Note: this is another example of substrate level
phosphorylation
1. Feed-forward regulation:
- In liver, pyruvate kinase is activated by F-1,6-BP, the
product of PFK reaction. This feed-forward regulation
has the effect of linking the 2 kinase activities:
increased PFK activity  elevated levels of F-1,6-BP
 activates pyruvate kinase
2. Covalent modulation of pyruvate kinase:
- Phosphorylation by a cAMP-dependent protein kinase
leads to inactivation of pyruvate kinase in the liver
- When blood gluc levels are low, elevated glucagon
increases intracellular level of cAMP  causes
phosphorylation & inactivation of pyruvate kinase.
Therefore, PEP is unable to continue in glycolysis, but
instead enters gluconeogenesis pathway.
- This, in part, explains the observed inhibition of hepatic
glycolysis & stimulation of gluconeogenesis by glucagon.
- Dephosphorylation of pyruvate kinase by a
phosphoprotein phosphatase results in reactivation of
enz
Figure 8.19
Covalent modification of pyruvate
kinase results in inactivation of
enzyme
3. Pyruvate kinase deficiency:
- Normal, mature RBC lacks mitochondria & is therefore,
completely dependent on glyolysis for production of ATP.
- ATP is required to meet metabolic needs of RBC, & also
to fuel the pumps necessary for maintenance of biconcave, flexible shape of the cell, which allows it to
squeeze through narrow capillaries
- Anemia observed in glycolytic enz deficiencies is a
consequence of reduced rate of glycolysis, leading to
decreased ATP production.
- Resulting alterations in RBC memb lead to changes in
shape of cell, and ultimately, to phagocytosis by cells of
reticuloendothelial system, particularly macrophages of
spleen
- Premature death & lysis of RBC  hemolytic anemia
- Among patients exhibiting genetic defects of glycolytic
enz’s, ~ 95% show a deficiency in pyruvate kinase, & 4%
exhibit phosphoglucose isomerase deficiency
- Pyruvate kinase (PK) deficiency is the 2nd most common
cause (after G-6PD deficiency) of enzymatic related
hemolytic anemia
- PK deficiency is restricted to RBCs, & produces mild to
severe chronic hemolytic anemia (RBC destruction), with
the severe form requiring regular cell transfusions
- Severity of disease depends both on degree of enz
deficiency (generally 5-25% of normal levels), & on
extent to which individual’s RBCs compensate by
synthesizing increased levels of 2,3-BPG
- Almost all individuals with PK deficiency have a mutant
enz that shows abnormal properties- most often altered
kinetics
Figure 8.20
Alterations observed with various
mutant forms of pyruvate kinase.
K. Reduction of pyruvate to lactate
Lactate, formed by action of lactate dehydrogenase, is
final product of anaerobic glycolysis in euk cells.
Formation of lactate is major fate of pyruvate in RBCs,
lens & cornea of the eye, kidney medulla, testes, &
leukocytes
1. Lactate formation in muscle:
In exercising skeletal muscle, NADH production (by
glyceraldehyde 3-P dehydrogenase & by the three
NAD+-linked dehydrogenases of TCA cycle) exceeds
oxidative capacity of respiratory chain
This results in elevated NADH/NAD+ ratio, favoring
reduction of pyruvate to lactate. Therefore, during
intense exercise, lactate accumulates in muscle,
causing a drop in intracellular pH, potentially resulting
in cramps.
Much of this lactate eventually diffuse into
bloodstream, & can be used by liver to make glucose
Figure 8.21. Interconversion of pyruvate and lactate.
2. Lactate consumption:
- The direction of lactate dehydrogenase reaction depends
on relative intracellular conc’s of pyruvate & lactate & on
ratio of NADH/NAD+ in the cell. E.g., in liver & heart,
ratio of NADH/NAD+ is lower than in exercising muscle.
These tissues oxidize lactate (obtained from blood) to
pyruvate
- In liver, pyruvate is either converted to glucose by
gluconeogenesis or oxidized in the TCA cycle
- Heart muscle exclusively oxidizes lactate to CO2 & H2O
via citric acid cycle
3. Lactic acidosis:
- Elevated conc’s of lactate in plasma termed
lactic acidosis occur when there is a collapse of
the circulatory system, e.g., in MI, pulmonary
embolism, & uncontrolled hemorrhage, or when
an individual is in shock
- Failure to bring adequate amounts of oxygen to
the tissues  impaired oxidative
phosphorylation & decreased ATP synthesis
- To survive, cells use anaerobic glycolysis as a
backup system for generating ATP, producing
lactic acid as the end-product
Note: production of even meager amounts of ATP
may be life-saving during the period required to
re-establish adequate blood flow to the tissues
- The excess oxygen required to recover from a
period when the availability of oxygen has been
inadequate is termed “oxygen debt”.
- The oxygen debt is often related to patient
morbidity or mortality.
- In many clinical situations, measuring the blood
levels of lactic acid provides for the rapid, early
detection of oxygen debt in patients.
- E.g., blood lactic acid levels can be used to
measure the presence & severity of shock, & to
monitor the patients’ recovery
L. Energy yield from glycolysis
-
Despite production of some ATP during
glycolysis, end-products, pyruvate or
lactate, still contain most of energy
originally contained in gluc. The TCA
cycle is required to release that energy
completely.
1. Anaerobic glycolysis:
- Two molecules of ATP are generated for
each molecule of gluc converted to 2
molecules of lactate. There is no net
production or consumption of NADH.
- Anaerobic glycolysis, although releasing
only a small fraction of energy contained in
gluc molecule, is a valuable source of
energy under several conditions, including:
1. when oxygen supply is limited, as in
muscle during intensive exercise
2. for tissues with few or no mitoch., such
as the medulla of the kidney, mature
RBCs, leukocytes, & cells of the lens,
cornea & testes
2. Aerobic glycolysis:
- Direct formation & consumption of ATP is the
same as in anerobic glycolysis i.e., a net gain of
2 ATP
- Two molecules of NADH are also produced per
molecule of gluc
- Ongoing aerobic glycolysis requires the
oxidation of most of this NADH by ETC,
producing ~ 3 ATP for each NADH entering the
chain (depending on the shuttle system)
Figure 8.22. Summary of anaerobic glycolysis. Reactions involving
the production or consumption of ATP or NADH are indicated. The
irreversible reactions of glycolysis are shown with thick arrows. DHAP
= dihydroxyacetone phosphate
VI. Hormonal regulation of glycolysis
- Regulation of glycolysis by allosteric activation or
inhibition, or phospho/dephospho of rate-limiting
enz’s, is short-term i.e., they influence gluc
consumption over periods of minutes or hours
- Superimposed on these moment-to-moment
effects are slower, often more profound,
hormonal influences on amount enz protein
synthesized. These effects can result in 10x to
20x fold increases in enz activity that typically
occur over hours to days
- Although current focus is on glycolysis, reciprocal
changes occur in the rate-limiting enz’s of
gluconeogenesis
- Regular consumption of meals rich in CHO or
administration of insulin initiates an increase in
the amount of glucokinase, PFK, & PK in liver
- These changes reflect an increase in gene
transcription, resulting in increased enz
synthesis.
- High activity of these 3 enz’s favors conversion
of gluc to pyruvate, a characteristic of the wellfed state
- Conversely, gene transcription and synthesis
glucokinase, PFK, & PK are decreased when
plasma glucagon is high & insulin is low, e.g., as
seen in fasting or diabetes
Figure 8.23
Effect of insulin and glucagon on
the synthesis of key enzymes of
glycolysis in liver.
VII. Alternate fates of pyruvate
A. Oxidative decarboxylation of pyruvate
-
-
Oxidative decarboxylation of pyruvate by pyruvate
dehydrogenase complex is an important pathway in
tissues with high oxidative capacity, e.g., cardiac
muscle.
Pyruvate dehydrogenase irreversibly converts
pyruvate, the end product of glycolysis, into acetyl
CoA, a a major fuel for TCA & the building block for
fatty acid synthesis
B. Carboxylation of pyruvate to oxaloacetate
- Carboxylation of pyruvate to OAA by pyruvate caroxylase
is a biotin-dependent reaction. This reaction is
important because it replenishes TCA cycle
intermediates, & provides substrate for
gluconeogenesis
Figure 8.24
Summary of the metabolic
fates of pyruvate.
C. Reduction of pyruvate to ethanol
(microorganisms)
- Conversion of pyruvate to ethanol occurs by two
reactions (Fig. 8-24)
- Decarboxylation of pyruvate by pyruvate
decarboxylase occurs in yeast & certain m/o’s,
but not in humans. The enz requires thiamine
pyrophosphate as a coenz, & catalyzes a
reaction similar to that described for pyruvate
dehydrogenase
Summary
• Most pathways can be classified as either catabolic
(degrade complex molecules to a few simple products)
or anabolic (synthesize complex end products from
simple precursors)
• Catabolic reactions also capture chemical energy in form
of ATP from degradation of energy-rich molecules
• Anabolic reactions require energy, which is generally
provided by breakdown of ATP
• Rate of a metabolic pathway can respond to regulatory
signals, e.g., allosteric activators or inhibitors, that arise
within the cell
• Signaling b/w cells provides for integration of metabolism
• The most important route of this communication is
chemical signaling b/w cells, e.g., by hormones or
neurotransmitters
• Second messenger molecules convey the intent of a
chemical signal (hormone or neurotransmitter) to
appropriate intracellular responders.
• Adenylyl cyclase is a memb-bound enz that syntheizes
cAMP in response to chemical signals, e.g., the
hormones glucagon & epinephrine
• Following binding to a hormone to its cell-surface
receptor, a GTP-dependent regulatory protein (Gprotein) is activated that, in turn, activates adenylyl
cyclase
• The cAMP activates a protein kinase, which
phosphorylates a cadre of enz’s, causing their activation
or deacivation/ ion channels regulation / gene expression
• Phospho is reversed by protein phosphatases
• Aerobic glycolysis, in which pyruvate is the end product,
occurs in cells with mitoch & an adequate supply of
oxygen
• Anaerobic glycolysis, in which lactic acid is the end
product, occurs in cells that lack mitoch, or in cells
deprived of sufficient oxygen
• Gluc is transported across memb’s by one of at least 14
glucose transporter isoforms (GLUTs). GLUT-1 is
abundant in RBCs & brain, GLUT-4 (which is insulindependent) is found in muscle & adipose tissue, &
GLUT-2 is found in liver & the β-cells of the pancreas
• Conversion of gluc to pyruvate (glycolysis) occurs in 2
stages: an energy investment phase in which phospho
intermediates are synthesized at expense of ATP, & an
energy generation phase, in which ATP is produced
• In energy investment phase, gluc is phosphorylated by
hexokinase (found in most tissues) or glucokinase (a
hexokinase found in liver cells & the β cells of pancreas)
• Hexokinase has a high affinity (low Km) & a small Vmax
for glucose, & is inhibited by G-6-P
• Glucokinase has a large Km & a large Vmax for glucose.
It is indirectly inhibited by F6P & activated by gluc, & the
transcription of the glucokinase gene is enhanced by
insulin.
• G6P is isomerized to F6P, which is phosphorylated to F1,6-BP by PFK. This enz is allosterically inhibited by ATP
& citrate, & activated by AMP.
• F-2,6-BP, whose synthesis is activated by insulin, is the
most potent allosteric activator of this enz. A total of 2
ATP are used during this phase of glycolysis.
• F-1,6-BP is cleaved  2 trioses further metabolized by
glycolytic pathway  pyruvate
• During these reactions, 4 ATP & 2 NADH are produced
from ADP & NAD+.
• Final step in pyruvate synthesis fro PEP is catalyzed by
PK. This enz is allosterically activated by F-1,6-BP, &
hormonally activated by insulin & inhibited by glucagon
via cAMP pathway.
• PK deficiency accounts for 95% of all inherited defects in
glycolytic enz’s. it is restricted to RBCs, & causes mild to
severe chronic hemolytic anemia.
• In anaerobic glycolysis, NADH is reoxidized to NAD+ by
conversion of pyruvate to lactic acid. This occurs in cells,
e.g., RBCs, that have few or no mitoch., & in tissues ,
such as exercising muscle, where production of NADH
exceeds oxidative capacity of respiratory chain
•
•
1.
2.
3.
Elevated conc’s of lactate in plasma (lactic acidosis)
occur when there is collapse of the circulatory system,
or when an individual in shock
Pyruvate can be:
Oxidatively decarboxylated by pyruvate
dehydrogenase, producing acetyl CoA
Carboxylated to OAA by pyruvate carobxylase
Reduced by m/o’s to ethanol by pyruvate
decarboxylase
Figure 8.25
Key concept
map for
glycolysis.