Download Lecture 22 - Introduction to Metabolism: Regulation Key Concepts

Bioc 460 - Dr. Miesfeld Fall 2008
Lecture 22 - Introduction to Metabolism: Regulation
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Key Concepts
• Six major groups of metabolic pathways
• Regulation of metabolic flux
• Glucagon, epinephrine, and insulin signaling pathways
KEY CONCEPT QUESTIONS:
What mechanisms control flux through metabolic pathways?
How do glucagon, epinephrine, and insulin control glucose levels?
Overview of metabolic pathways
Small biomolecules serve as metabolites (reactants and products) in biochemical reactions within
cells that are required for life-sustaining processes.
Enzymes (either protein or RNA) are the chemical catalysts
Figure 1.
that drive these biochemical reactions. In these enzymemediated biochemical reactions, the products of one
reaction are inevitably the reactants for other functionallyrelated reactions. The thousands of reactions in a cell
required for sustaining life are interdependent and highly
regulated to maximize efficient use of limiting metabolic
resources.
The emerging discipline of systems biology
attempts to describe complex chemical reaction networks in
cells using mathematical models that are able to predict
metabolic flux (reactant and product concentrations over time) in response to environmental or
physiological conditions. Systems biology can provide a better paradigm for presenting a global
picture of cellular metabolism, it is more instructive to focus on a limited number of biochemical
reactions that have been highly characterized and which provide a basis for understanding the
chemistry of life. Using this approach, sets of
Figure 2.
biochemical reactions are grouped together into
metabolic pathways (figure 1 ).
In order to understand how a metabolic pathway
is organized, let's first look at a mini-pathway
consisting of just two reactions. As shown in figure 2,
the reactants aspartate and citrulline are metabolites in
the urea cycle which is a metabolic pathway in liver
cells responsible for nitrogen excretion in animals. The
enzyme argininosuccinate synthase is a protein that
catalyzes a condensation reaction that forms the
product argininosuccinate using phosphoryl transfer
energy made available by ATP. In the second
reaction, argininosuccinate is cleaved by the enzyme
argininosuccinate lyase to form the products
fumarate and arginine. The chemical difference
between citrulline and asparagine is the addition of a
single amino group obtained from aspartate, however
in order for this to occur, argininosuccinate has to function both as a product and a reactant.
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An example of a more complex metabolic pathway is
Figure 3.
illustrated in figure 3 where it can be seen that enzymes
interconvert metabolites using reversible and irreversible
reactions. In some reactions, phosphoryl bond energy
available in ATP is used to drive the reaction toward product
formation. Three types of linked reactions are commonly found
in metabolism. The most common type is linear pathways in
which each reaction generates only a single product that is a
reactant for the next reaction in the pathway. In contrast,
forked pathways usually generate two products, each of which
as a different metabolic fate. Lastly, cyclic pathways contain a number of metabolites that are
regenerated during each turn of the cycle, serving as both reactants and products in every
reaction. Two examples of cyclic pathways we will discuss later in the course are the citrate cycle
and the urea cycle. Note that the flux of metabolites through metabolic pathways is dependent on
both the activity of each
Figure 4.
enzyme in the pathway, and
the intracellular
concentrations of reactants
and products which affects
the directionality of reactions
as a function of mass action
(Le Chatelier's principle).
Figure 4 shows the
basic metabolic map we will
use in this course which is
designed to highlight the
interdependence of six major
groups of pathways (both
anabolic and catabolic). A
full page copy of this
metabolic map can be
downloaded from the course
website. The layout of
pathways in this version of a
metabolic map was designed
to illustrates the hierarchical
nature of metabolism which
includes four classes of
macromolecules (proteins,
nucleic acids, carbohydrates,
and lipids), six primary
metabolites (amino acids,
nucleotides, fatty acids,
glucose, pyruvate, acetyl
CoA) and seven small
biomolecules (NH4+, CO2,
NADH, FADH2, O2, ATP,
H2O), all of which we will encounter frequently on our journey. The metabolic chart in figure 4 will
be used as a template each time a new pathway is introduced using the “divide and conquer”
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strategy illustrated in figure 5. We will start by describing
Figure 5.
pathways involved in energy conversion processes
(glycolysis, citrate cycle, electron transport system,
oxidative phosphorylation, photophosphorylation, and
carbon fixation), and then survey both degradative and
biosynthetic pathways in carbohydrate metabolism
(pentose phosphate pathway, gluconeogenesis, glycogen
degradation and synthesis), lipid metabolism (fatty acid
oxidation and synthesis, lipoprotein functions, steroid and
eicosanoid synthesis), and amino acid metabolism (amino
acid degradation and synthesis). Figure 6 summarizes the
road map we will use for these lectures. The boundaries
we have drawn to separate the six major groups of
pathways are somewhat arbitrary. Nevertheless, I have
chosen this particular pedagogical strategy for two
reasons, 1) it underscores the importance of energy
conversion as the foundation for all other metabolic
pathways; without sunlight and the fuel produced by
photosynthetic organisms, life would not exist on this
planet as we know it, and 2) it permits us to maintain our
focus on the role of proteins in biochemical processes, in
this case, enzyme regulation of metabolic flux through linked reactions in a pathway.
We will start off the discussion of major pathways in metabolism by answering the following
four questions:
1. What does the pathway accomplish for the cell?
2. What is the overall net reaction of the pathway?
3. What are the key enzymes in the pathway?
4. What are examples of this pathway in real life?
Figure 6.
The answers to these four simple questions provides you
with the basic information you will need to study the ten
individual pathways we will cover as shown below:
Pathway(s)
Glycolysis
Citrate Cycle
Oxidative Phosphorylation
Photosynthesis
Pentose Phosphate Pathway
Gluconeogenesis
Glycogen Degradation and Synthesis
Fatty Acid Degradation and Synthesis
Nitrogen fixation and assimilation
Urea Cycle
Lectures
23, 24
25, 26
27, 28
29, 30
32
32
33
34, 35
37
37
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Regulation of metabolic flux
As described in lecture 21, catabolic pathways are defined as the collection of enzymatic
reactions in the cell that lead to the degradation of macromolecules and nutrients for the purpose
of energy capture, usually in the form of ATP and reducing power (NADH and FADH2). In contrast,
anabolic pathways utilize energy available from the hydrolysis of ATP and the oxidation of
reducing equivalents (primarily NADPH) to synthesize biomolecules for the cell. Importantly,
catabolic and anabolic pathways are active at the same time in the cell and many metabolites
serve as both substrates and products for different enzymes. Sometimes the catabolic pathways
are more active than the opposing anabolic pathway, whereas at other times, anabolic pathways
predominate when energy stores are plentiful and the necessary building blocks are available.
The flow or flux of metabolites through catabolic and anabolic pathways is determined by
two primary factors, 1) availability of substrates (diet or stored reserves) and 2) level of enzyme
activity which is controlled by enzyme levels (gene transcription and protein synthesis), catalytic
activity (allosteric control and covalent modification), and compartmentation (subcellular or tissue
selective localization). As was described in the first half of the course, enzymes function by
providing a suitable reaction environment (the enzyme active site) that serves to lower the energy
of activation for a given reaction. Enzymes cannot change the equilibrium of a reaction, but
instead, function as catalysts that increase reaction rates. With this in mind, reversible reactions in
metabolism normally function near equilibrium and are therefore controlled by substrate
availability, whereas, irreversible reactions are catalyzed by highly-regulated enzymes that function
far from equilibrium. One of the best ways to understand how flux through various catabolic and
anabolic pathways changes in response to substrate concentration and enzyme activity levels is to
look at glucose metabolism in the liver before and after breakfast. The two primary hormones
that control serum glucose levels are glucagon, the "I am hungry" hormone, and insulin, the "I
just at ate" hormone. A third hormone that controls glucose levels in the blood is epinephrine
(adrenaline), which I call the "I just saw a snake" hormone. Epinephrine signaling through β2
adrenergic receptors in liver cells also leads to the release of glucose by a similar mechanism.
As illustrated in figure 7, in the morning before your first meal, blood glucose levels begin to
decline after a night of “fasting” which triggers glucagon release from the pancreas. Glucagon
signaling in liver cells activates both a catabolic pathway (glycogen degradation) and an anabolic
pathway (gluconeogenesis), while at the same time inhibiting the catabolism of glucose by the
glycolytic pathway. However, within an hour of eating a bowl of cereal and drinking a cup of fruit
Figure 7.
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juice, your insulin levels increase due to elevated
blood glucose causing activation of the insulin
signaling pathway and stimulation of glucose uptake,
glycogen synthesis, and an increase in glucose
catabolism by the glycolytic pathway. Therefore, by
“breaking your fast” you initiate a transient shift in flux
through these various metabolic pathways until blood
glucose levels stabilize around 5 mM. Importantly,
the four pathways shown in figure 7 are active in liver
cells all of the time, with the only change being the
relative metabolite flux through each pathway in
response to glucose concentrations and hormone
activation of key enzymes.
Figure 8.
Glucagon, epinephrine, and insulin signaling
Since glucagon, epinephrine, and insulin signaling
will be used numerous times as examples of
metabolic regulation during this half of the course, we
need to take a closer look at how these two peptide
hormones transmit intracellular signals. Glucagon
signals through a G protein-coupled receptor pathway that activates protein kinase A (PKA) as
a result of increased levels of the second messenger cyclic AMP. In contrast, insulin hormone
signals through a receptor tyrosine kinase mechanism involving activation of phosphoinositol-3
kinase (PI-3K). Importantly, glucagon and
Figure 9.
epinephrine signaling (figure 8), and insulin
signaling (figure 9), control glucose levels in
the blood by regulating glycogen metabolism
and gluconeogenesis. Note that insulin
signaling also activates a separate pathway
involving the small G protein Ras which
controls cell proliferation.
Glucagon is released by the pancreas
and has been called the hunger hormone
because it signals low blood glucose levels.
Consistent with this physiological role,
glucagon receptors are primarily expressed
in liver and adipocyte cells where energy
stores in the form of glycogen and fatty acid
are located. Epinephrine-induced glycogen
breakdown in muscle cells produces glucose
as source of chemical energy to generate ATP which is needed for muscle contraction. Regulation
of glycogen breakdown in liver cells by epinephrine and glucagon is dependent on the presence of
β2 adrenergic and glucagon receptors, respectively. The biochemistry of these two signaling
mechanisms is essentially identical with both receptor systems utilizing the same Gsα signaling
pathway to stimulate adenylate cyclase (figure 8). Keep in mind that the physiological stimuli
responsible for epinephrine secretion by the adrenal medulla (fight or flight response), and
glucagon release from the pancreas (low blood glucose levels), are somewhat distinct and
therefore these pathways are not usually stimulated in liver cells at the same time.
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As shown in figure 10, stimulation of adenylate cyclase activity by activated Gsα, leads to
production of cAMP
and subsequent
Figure 10.
dissociation of the
PKA regulatory and
catalytic subunits.
As described in
more detail in
lecture 33, the two
key regulatory
enzymes in
glycogen
metabolism are
phosphorylase
kinase and
glycogen
synthase, both of
which are
phosphorylated in
liver cells by PKA.
Phosphorylation of
phosphorylase
kinase by PKA is
an activating signal
that transmits the
epinephrine signal
downstream
through
phosphorylation of
its target protein
glycogen
phosphorylase.
Glycogen phosphorylase catalyzes the removal of glucose units from glycogen through a
phosphorolysis reaction (lecture 33). In contrast, PKA phosphorylation of glycogen synthase is an
inhibitory signal that reduces enzyme activity and leads to a decrease in glycogen biosynthesis.
The net result of PKA-mediated phosphorylation of phosphorylase kinase and glycogen synthase
is increased glucose export to peripheral tissues.
In summary, activation of the epinephrine or glucagon G protein-coupled receptors in liver
cells results in increased intracellular levels of cAMP which initiates a PKA-dependent
phosphorylation cascade culminating in glucose export. Importantly, the phosphorylation cascade
involves both the activation and inhibition of downstream target proteins, that together, provide a
rapid response to external stimuli (fear or hunger).
The insulin receptor is synthesized as a single polypeptide chain that is processed by
proteolytic cleavage to produce a disulfide-linked protein. The mature insulin receptor is an α2β2
heterotetramer consisting of cross-linked α and β chains connected by disulfide bridges. The α
subunits form the extracellular ligand binding domain and the β subunits encode the intracellular
protein tyrosine kinase function. Insulin binding to α subunits causes a conformational change in
the receptor that stimulates the protein tyrosine kinase activity in the β subunits, resulting in
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receptor
Figure 11.
autophosphorylation.
Activation of insulin
receptor tyrosine kinase
activity leads to tyrosine
phosphorylation of a family
of proteins called insulin
receptor substrate (IRS)
proteins that bind to the β
subunits (figure 11).
Recruitment of PI-3 kinase
to the plasma membrane by
IRS proteins stimulates
phosphorylation of
phosphatidylinositol 4,5bisphosphate (PIP2) to
produce
phosphatidylinositol
3,4,5-triphosphate (PIP3)
which remains in the
plasma membrane and
serves as a docking site for
signaling proteins
containing pleckstrin
homology (PH) domains.
PH domains are highly
conserved protein structural
motifs that serve to recruit
signaling proteins to the
plasma membrane.
Two proteins in the
insulin signaling pathway
containing PH domains are
phosphoinositol-dependent kinase (PDK1), and Akt, a serine-threonine kinase originally
identified as an oncogene gene (cancer gene) in the Akt8 murine retrovirus. Akt is also known as
protein kinase B (PKB) because it shares amino acid homology with protein kinase A and protein
kinase C. Both PDK1 and Akt bind to PIP3 leading to PDK1 phosphorylation of Akt and
subsequent dissociation of activated Akt from PIP3. Akt diffuses through the cytosol where it
phosphorylates numerous downstream targets. The net result is glucose uptake and increased
glycogen synthesis to store the glucose for use later. Insulin signaling is similar in liver, muscle,
and adipose tissue.
Since glycogen phosphorylase and glycogen synthase have opposing effects on glycogen
metabolism, it is critical that their activities be reciprocally regulated to avoid futile cycling and to
efficiently control glucose-6P concentrations within the cell. Figure 12 summarizes the effects of
glucagon and insulin signaling on glycogen metabolism in liver cells where it can be seen that
glucagon stimulates glucose efflux and insulin stimulates glucose influx through the GLUT2
glucose transporter protein. It can be seen that net phosphorylation drives glycogen degradation
and net dephosphorylation drives glycogen synthesis.
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ANSWERS TO KEY CONCEPT QUESTIONS:
Figure 17.
What mechanisms control flux through
metabolic pathways?
Substrate availability and enzyme activity
levels control flux through metabolic
pathways. Substrate availability is
determined by both diet and release of
stored metabolites, primarily glucose and
fatty acids, although amino acids can also
become available from increased rates of
protein degradation. Enzyme activities are
controlled by enzyme levels (gene
expression protein synthesis, and rates of
protein turnover), catalytic activity (allosteric
control and covalent modification), and by
compartmentation (subcellular organelles
and tissue-selective localization). Changes
in diet or physical activity affect the rate of
metabolic flux in animals, whereas,
environmental changes such as
temperature, water, or sunlight, affect the
rate of metabolic flux in plants and bacteria.
The most highly regulated enzymes in
metabolic pathways are those that control rate-limiting reactions, or catalyze the committed step in
a pathway. The committed step in the pathway is one in which the product of the reaction has only
one metabolic fate, and is thereby committed to be metabolized by the next enzyme in the
pathway.
How do glucagon, epinephrine, and insulin control glucose levels?
Glucagon, epinephrine, and insulin are hormones that bind to membrane receptors on target cells
and activate intracellular signaling pathways. Glucagon and epinephrine (adrenaline) signal the
blood glucose levels are low (glucagon), or that glucose is needed quickly for muscle contraction
(epinephrine). Both glucagon and epinephrine bind to G protein coupled receptors that activate
second messenger-dependent pathways. Glucagon receptors stimulate Gsα proteins which
activate adenylylate cyclase and produce high intracellular levels of cyclic AMP (cAMP).
Epinephrine binding to β2 adrenergic receptors in liver cells also activates Gsα signaling, however,
epinephrine binding to α1 adrenergic receptors in muscle cells activates phospholipase C
signaling. cAMP activation of protein kinase A (PKA) activity leads to the phosphorylation of
downstream target proteins, which together, lead to the accumulation of glucose through activation
of both glycogen degradation and gluconeogenesis. Increased insulin levels signals to cells that
glucose is plentiful and it should be metabolized to generate ATP and also stored for later use by
building up glycogen levels. Insulin binds to a receptor tyrosine kinase on target cells and initiates
a signaling cascade in involving the enzyme phosphoinositol-3 kinase (PI-3K) which
phosphorylates another kinase called AKT which controls glucose metabolism through several
mechanisms. Insulin activates glucose important and glycogen synthesis.
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