Download File

Introduction to Metabolism
Chapter 8
Metabolism is the set of life-sustaining chemical transformations
within the cells of living organisms. These enzyme-catalyzed reactions
allow organisms to grow and reproduce, maintain their structures, and
respond to their environments.
1a. Using graphs and words, compare and
contrast Exergonic vs. Endergonic chemical
reactions
Exergonic reactions are those that release energy. During these reaction the
reactant has higher free energy than the product. This is expressed by -G
(negative Gibbs number). These reaction occurs spontaneously and their rate is
usually increased by enzymes.
An example is the breakdown of ATP into inorganic phosphate ion and ADP during
exerting work.
ATP----> ADP + Pi+ energy
Endergonic reactions are those that store energy. During these reactions the
reactant has lower free energy than the product. This is expressed by +G (positive
Gibbs number).
They do not happen spontaneously and need supply of energy to occur. Example
would be production of ATP during aerobic respiration:
ADP+ Pi + energy -----> ATP
Because one reaction releases and one requires energy, many times reactions
that are endergonic and exergonic are coupled together (such as redox reactions)
An exothermic reaction can be either endergonic or exergonic. The same is also
true for an endothermic reaction, as in your example. You'd have delta H > 0
(endothermic) but delta G < 0 (exergonic).
Exergonic vs. Endergonic
• More energy in
reactants than
products.
• Delta G negative
• Energy is released
• Catabolic
• Spontaneous
• Increased stability
(increased entropy)
• More energy in
products than
reactants.
• Delta G positive
• Energy is added
• Anabolic
• Non-spontaneous
• Decreased stability
(decreased entropy)
1b. Chemical reactions with
and without an enzyme
With an enzyme:
• Energy of activation is lowered
• Speed of reaction is faster
• Energy in products and reactants are
unchanged
• The sign of delta G does not change.
• A non-spontaneous reaction does not
become spontaneous.
2. Explain how cells are able to remain alive and
increase in complexity in accordance with the
second law of thermodynamics
• 2nd law: Disorder (entropy) in the universe is
continuously increasing.
• In general, energy transformations proceed
spontaneously to convert matter from a more ordered,
less stable form, to a less ordered, more stable form.
• To remain alive, delta G cannot be equal to 0. This is
done by coupling endergonic chemical reactions that
increase or maintain complexity to exergonic chemical
reactions that release energy. Product becomes reactant
for the next chemical reaction
• . Heterotrophic organisms breakdown
organic compounds (chemical bonds
within compounds are sources of potential
energy). Autotrophic organisms convert
one form of energy (photons of light or
energy in inorganic molecules) to another
form: chemical bonds in organic molecules
(again, a source of potential energystored).
3. Compare the strategies employed by
different lineages of cells to acquire and
utilize free energy
• Autotrophs: Photoautotrophs that convert sunlight into
chemical energy (acquire); chemoautotrophs that
convert inorganic compounds into organic molecules
(acquire). Stored in new chemical bonds. Released to
do work.
• Heterotrophs: Breakdown ingested organic compounds
(acquire)via aerobic cell respiration. Herbivores vs.
carnivores vs. decomposers
• Heterotrophs: Breakdown ingested or absorbed
(acquired) organic compounds via anaerobic cell
respiration/fermentation.
• Utilize this acquired free energy to power cellular
activities: transport/movement /chemical reactions.
4. How does ATP perform work?
Composition of ATP
a) 5 carbon sugar –
ribose
b) adenine (purine)
c) triphosphate
group: PO4- x 3
ATP
• A) negative charges of
phosphate groups repulse one
another in a tight area. This
causes
• B) 2 covalent bonds between
phosphate groups to be
unstable. Coiled spring
analogy: phosphates straining
away from one another.
• C) These 2 bonds have low
activation energy and are
easily broken through
hydrolysis.
• Only outermost high
energy phosphate bond is
hydrolyzed, cleaving off
phosphate group at the
end.
• ATP + water = ADP
(adenosine diphosphate)
+ inorganic phosphate
• In a test tube, this
reaction generates a
change in free energy of
– 7.3 kcal/mole
• In a cellular environment,
delta G = -13 kcal/mole
ATP hydrolysis: coupling by phosphate transfer E.G. Glutamic Acid +
Ammonia to Glutamine
• ATP hydrolysis is coupled
to an endergonic process
(energy requiring) by
transferring a phosphate
group from ATP to some
other molecule.
• Phosphorylation
produces a
phosphorylated
intermediate.
• This intermediate is less
stable than original
molecule. This is energy
coupling by phosphate
transfer.
5. Using graphs and words, explain the effects of
temperature and pH on enzyme activity
a)
Temperature: Up to a point,
increased temperature
increases the rate of a
chemical reaction by
increasing the number of
collisions between enzyme
and substrate.
At some point, increased
temperature stops chemical
reaction by denaturing
protein by disrupting ionic, H
bonds, weak interactions.
Each enzyme has an optimum
range of temperatures in
which to function.
pH
• Each enzyme has an
optimum pH range
usually between 6 –
8. Exception: pepsin
which begins the
digestion of protein in
the mammalian
stomach. Optimal pH
is 2.0
• A pH that is out of the
range may denature
an enzyme, causing it
to lose it’s secondary
and tertiary structures
and resulting in the
loss of activity.
6. Using a graph and words, how does increased
substrate concentration affect enzyme activity?
Rate of Conversion of Substrate
• Partly dependent on initial concentration of substrate.
Increased concentration, increases the reaction rate
because of the increased chance of contact.
• Substrate concentration can affect the rate of reaction
until all of the active sites are filled (saturated).
Rate of
Reaction
Substrate Concentration
Saturation
Examples of Competitive and
non-competitive inhibition
• An inhibiting molecule
structurally similar to the
substrate molecule binds to the
active site, preventing
substrate binding. Eg.
Inhibition of folic acid synthesis
in bacteria by the sulfonamide
(antibiotic) Prontosil. E.g.
Carbon monoxide binds to the
active site of hemoglobin and
is a competitive inhibitor that
binds irreversibly.
• An inhibitor molecule binding
to an enzyme (not to its active
site) that causes a
conformational change in its
active site resulting in a
decrease in activity. Inhibitor
does not have to be chemically
similar to substrate.
• Eg. Metal ions disrupting
disulfide bridges in many
enzymes including cytochrome
oxidase (enzyme in electron
transport chain). Hg2+, Ag+,
Cu2+ bind to –SH groups,
breaking –S-S- linkages;
changes shape of the active
site.
7. Competitive vs. Noncompetitive inhibitors
• Competitive: chemically
similar; competes with
substrate for active site. Effect
can be diluted with addition of
substrate.
• Non-competitive: does not
have to be chemically similar.
Binds somewhere else on
enzyme that is not the active
site. Changes the active site
so that it does not bind
substrate in the way that it did
without an inhibitor.
8.
Explain allosteric regulation of enzymes as an
example of feedback inhibition (negative feedback)
• A particular type of noncompetitive inhibition of
biochemical pathways
• End-product binds to first
enzyme in pathway and shuts
the pathway down.
• Also called: End-product
inhibition; a version of negative
feedback
• Mechanism of homeostasis
• The allosteric site is a
specific portion of an
enzyme. Not the
active site.
• Serves as an on-off
switch. Binding of a
substance to this site
can switch an enzyme
between active and
inactive configuration.
2 Examples of allosteric enzymes:
phosphofructokinase and threonine deaminase
• Pathway is switched off by its
end-product which acts as an
inhibitor.
• This is a form of noncompetitive inhibition.
• Shape of the allosteric enzyme
can be altered by the binding
of end products to an allosteric
site, decreasing its activity.
• E.g. ATP inhibition of
phosphofructokinase in
glycolysis where ATP turns off
its own production.
Other things to worry about:
• Models of enzyme
specificity
• Equation for free
energy
• Types of work that
cells do
• Examples of how
active sites are
involved in catalysis.
• Use of enzymes in
biotechnology
• E.g. Lactase,
pectinase, proteases
1. METABOLISM
Definition: all of the 1000s of precisely coordinated,
complex, efficient and integrated chemical reactions in
an organism.
Metabolic pathways: chemical reactions ordered into
sequenced branching routes controlled by enzymes.
a) Metabolic pathways that release energy by breaking
down complex molecules to simpler molecules:
CATABOLIC PATHWAYS.
E.G. Cellular respiration where glucose and other
organic fuels broken down to carbon dioxide and water;
energy is released to do work.
Metabolic pathways continued
b) Metabolic pathways which consume
energy to build complex molecules from
simpler ones: Anabolic Pathways.
e.g. Synthesis of proteins from amino
acids (for defense, motion, transport).
Common to Both Pathways: the
Involvement of Energy
• Energy coupling: energy released from
catabolic pathways fuel anabolic
pathways.
2. Bioenergetics: analysis of how
energy powers the activities of
living organisms
Energy: the capacity to do work
Energy exists in 2 states:
a) Kinetic energy: energy of motion.
Moving objects perform work by causing
other matter to move.
e.g. Flow of electrons: electricity; nerve
which transmits signals; lightphotosynthesis
KINETIC ENERGY
b) Potential energy: stored
energy. Not moving but having the
capacity to move
• The energy of position
c. Examples of energy forms
• Mechanical: mechanoreceptors =
pressure, touch
• Heat: thermal receptors
• Sound: also a mechanoreceptor
• Electrical current
• Light: photoreceptors (eye)
• Radioactive radiation
• Chemical: chemoreceptors – taste, smell
d) All forms of energy can be
converted to heat
• We can express energy in terms of heat.
• The study of energy: thermodynamics
(heat change).
• Unit of heat most commonly used in
biology: kilocalorie (1000 calories).
• Unit of heat in Physics is the joule =
0.239cal.
e) Energy flow into the biological
world: the sun
• In photosynthesis: small molecules
combine into more complex molecules.
Energy is stored as potential energy in the
covalent bonds between atoms in the
sugar molecules.
f) During a chemical reaction, the
energy stored in chemical bonds
may transfer to new bonds
Electrons may pass from one atom or molecule
to another.
Loss of electrons: oxidized
Gain of electrons: reduced
These reactions always take place together:
oxidation-reduction reactions (redox).
Redox reactions play a key role in the flow of
energy through biological systems because the
electrons that pass from one atom to another
carry energy with them.
• If electron is boosted into another energy
level (light) the electron’s extra energy is
transferred with it. Added energy is stored
as potential energy that the atom can later
release when the electron returns to its
original energy level.
3. The Laws of Thermodynamics
A set of universal laws that govern all
energy changes in the universe, from
nuclear reactions to the buzzing of a bee.
a) The 1st law of thermodynamics:
concerns the amount of energy in the
universe. Energy can change from one
form to another, but can never be
destroyed nor created.
1st Law
• Potential energy can be converted to
kinetic energy.
• During energy conversion, some energy is
lost as heat.
• Energy flow in the biological world is
unidirectional:
• Sun
energy enters system and
replaces energy lost from heat
b) 2nd Law of Thermodynamics
• Concerns the transformation of potential
energy to heat, or random molecular
motion
• Disorder (entropy) in the universe is
continuously increasing.
• In general, energy transformations
proceed spontaneously to convert matter
from a more ordered, less stable form, to a
less ordered, more stable form.
c. Entropy: measure of the
disorder of a system
d) Free energy: the amount of energy actually
available to break and form other chemical
bonds…energy available to do work.
G = (free energy) after Gibbs
H = energy contained in a molecule’s chemical
bonds (enthalpy)
S = Entropy (the energy unavailable because of
disorder)
T = Temperature in degrees Kelvin
Free Energy Equation
• G = H – TS
• Change in free energy (delta G): Change
in H – T x change in S.
• Change in free energy positive number:
products of reaction has more energy than
reactants. Endergonic chemical reaction
(energy requiring) because either H has
increased and/or S has decreased.
Change in Free energy: Negative
• Change in G negative number: products
of reaction have less energy than
reactants. Exergonic (energy liberating)
chemical reaction. Spontaneous
Free Energy characteristics
Exergonic vs. Endergonic
e) Activation Energy
• Required energy input to begin a chemical
reaction, even if chemical reaction is
spontaneous.
• Catalysis: process of influencing chemical
bonds in a way that lowers the activation
energy needed to initiate a reaction.
• Catalysts speed up the process of
chemical reactions without being changed
by the reaction.
With and Without Enzyme
4. Catalysts
• A) do not violate the laws of thermodynamics.
Endergonic reaction will not occur
spontaneously.
• B) decrease activation energy
• C) accelerate both forward and reverse
reactions by exactly the same amount.
• D) do not determine the direction of the chemical
reaction which is determined solely by the
difference in free energy. Does not alter
proportions of reactants converted to products.
• E) Do not change which chemical reactions are
spontaneous.
Summary: Exergonic vs.
Endergonic Reactions
1. Net release of free
energy
2. Delta G = -number
3. Can be spontaneous
4. E.g. cell respiration
5. E.g. 1 mole of
glucose contains
Delta G = -686
Kcal/mol
1. Absorbs free energy
from surroundings
2. Delta G = + number
3. Nonspontaneous
4. E.g. photosynthesis
5. Eg. CO2 + H20
glucose
Delta G = +686Kcal/mol
More Endergonic vs. Exergonic
If a reversible process is endergonic in one
direction, reverse process is exergonic
At equilibrium, the change in free energy = 0
As a result you can do no work; dead!
In living cells, delta G is never allowed to reach 0
Product of 1 reaction becomes the reactant in
another reaction on down the pathway.
Cell Respiration is driven by: great difference
between the free energy of glucose and the free
energy of carbon dioxide and water.
Hydroelectric Analogy
5. ATP
• Chief energy currency that all cells use;
most of the energy harvested in plants is
used to manufacture ATP.
• ATP = Adenosine triphosphate
• A)Composition of ATP
a) 5 carbon sugar – ribose
b) adenine (purine)
c) triphosphate group: PO4- x 3
ATP Structure
More on the triphosphate group
• A) negative charges of phosphate groups
repulse one another in a tight area. This causes
• B) 2 covalent bonds between phosphate groups
to be unstable. Coiled spring analogy:
phosphates straining away from one another.
• C) These 2 bonds have low activation energy
and are easily broken through hydrolysis.
b) Reactions involving ATP:
• Only outermost high energy phosphate
bond is hydrolyzed, cleaving off phosphate
group at the end.
• ATP + water
ADP (adenosine
diphosphate) + inorganic phosphate
• In a test tube, this reaction generates a
change in free energy of – 7.3 kcal/mole
• In a cellular environment, delta G = -13
kcal/mole
c) ATP hydrolysis is coupled
• ATP hydrolysis is coupled to an
endergonic process (energy requiring) by
transferring a phosphate group from ATP
to some other molecule.
• Phosphorylation produces a
phosphorylated intermediate.
• This intermediate is less stable than
original molecule. This is energy coupling
by phosphate transfer.
ATP Hydrolysis: Unstable
Phosphorylated Intermediate
Example: Glutamic Acid to
Glutamine
• The addition of ammonia to glutamic acid makes
a different amino acid = glutamine
• This process is an endergonic process and
requires energy.
• Delta G = +3.4 Kcal/mole
• It is coupled to the phosphorylation of glutamic
acid by ATP, which transfers chemical instability
to the amino acid.
• 2nd step: ammonia displaces the phosphate
group from the intermediate, forming glutamine.
• ATP is regenerated
Regeneration of ATP
• Involves the conversion of ADP and
inorganic phosphate to ATP and water
• Energy requiring process that is coupled to
an exergonic pathway: cellular respiration.
• Let’s review an energy profile of a
reaction
ATP Cycle
Exergonic Chemical Reaction
Enzyme Activity: A Closer Look
a) Enzymes are substrate specific
b) Definition of substrate: reactant upon which an
enzyme acts
c) Enzymes convert reactants to products by
joining with reactants or substrates
d) Enzymes can distinguish its substrate from
closely related compounds such as isomers.
e) The part of the enzyme that binds to substrate
is called the active site
The Active Site
a) May be only a few amino acids long
b) May be a pocket or groove on the
surface of a protein
c) Enzyme specificity is based on its shape.
Models of Enzyme-Substrate
Specificity
Lock and Key
Fit between the shape and
chemistry of its active site
and the shape of the
substrate described as
lock (enzyme) and key
(substrate).
Implies rigidity. Shape is
not flexible.
Each enzyme only binds to
one substrate.
Induced Fit: more like a
handshake. Active site is
rigid; as substrate enters
the active site, it is
induced to change shape
by the substrate. Result:
active site fits even more
snugly around the
substrate. An enzyme
might bind >1 substrate.
Accounts for the broad
specificity of some
enzymes.
ENZYME INDUCED FIT
Advantage of induced fit model
• Induced fit brings chemical groups of the
active site into positions that enhance their
ability to work on the substrate and
catalyze the chemical reactions.
The Active Site: Possible Modes of
Action
1.
2.
3.
4.
5.
Holds substrate using hydrogen and ionic bonds; weak
interactions.
R groups of a few amino acids in active site catalyze
conversion of substrate to product. Remember that
enzyme is not changed by the reaction.
Able to convert more than a thousand molecules per
second, some enzymes are faster.
In some cases, active site provides a template for the
substrates to come together. It may align the substrate
so that the substrate can interact with the template.
Active site might be a pocket of low or high pH
CATALYTIC CYCLE: Sucrase
Environmental Factors that Affect
Enzyme Activity
a) Temperature: Up to a point, increased
temperature increases the rate of a chemical
reaction by increasing the number of collisions
between enzyme and substrate.
At some point, increased temperature stops
chemical reaction by denaturing protein by
disrupting ionic, H bonds, weak interactions.
Each enzyme has an optimum range of
temperatures in which to function.
ENZYME REGULATION
b) pH
• Each enzyme has an optimum pH range
usually between 6 – 8. Exception: pepsin
which begins the digestion of protein in the
mammalian stomach. Optimal pH is 2.0
Speed of a chemical reaction
• Determined by the speed at which the
active site can convert substrate to
product or can manufacture more enzyme.
c) Inhibitors
• Chemicals other than intended reactant
bonded to the active site or changing the
shape of the active site.
• Two general types: Competitive and
Noncompetitive
Competitive and Noncompetitive
Inhibitors
Competitive Inhibitors
• An inhibiting molecule structurally similar
to the substrate molecule binds to the
active site, preventing substrate binding.
Eg. Inhibition of folic acid synthesis in
bacteria by the sulfonamide (antibiotic)
Prontosil. E.g. Carbon monoxide binds to
the active site of hemoglobin and is a
competitive inhibitor that binds irreversibly.
Non-Competitive Inhibition
• An inhibitor molecule binding to an enzyme (not
to its active site) that causes a conformational
change in its active site resulting in a decrease
in activity. Inhibitor does not have to be
chemically similar to substrate.
• Eg. Metal ions disrupting disulfide bridges in
many enzymes including cytochrome oxidase
(enzyme in electron transport chain). Hg2+, Ag+,
Cu2+ bind to –SH groups, breaking –S-Slinkages; changes shape of the active site.
Allosteric Inhibition
• A particular type of non-competitive
inhibition of biochemical pathways
• End-product binds to first enzyme in
pathway and shuts the pathway down.
• Also called: End-product inhibition; a
version of negative feedback
• Mechanism of homeostasis
Allostery and control of metabolic
pathways by end-product inhibition
• The allosteric site is a specific portion of an
enzyme. Not the active site.
• Serves as an on-off switch. Binding of a
substance to this site can switch an enzyme
between active and inactive configuration.
• If a substance decreases its protein’s activity:
allosteric inhibitor
• If a substance increases its protein’s activity:
allosteric activator
• When an active site is stabilized by a substrate
molecule: exhibits cooperativity.
ALLOSTERIC REGULATION
COOPERATIVITY
Feedback Inhibition
• Pathway is switched off by its end-product
which acts as an inhibitor.
• This is a form of non-competitive inhibition.
• Shape of the allosteric enzyme can be
altered by the binding of end products to
an allosteric site, decreasing its activity.
• E.g. ATP inhibition of phosphofructokinase
in glycolysis where ATP turns off its own
production.
E.g. Pathway that converts
threonine to isoleucine
• This pathway shuts down when isoleucine
accumulates.
• Isoleucine is an allosteric inhibitor of the
enzyme that catalyzes the first step in the
pathway.
END PRODUCT INHIBITION
Location of Enzymes
• Some enzymes have defined locations (e.g.
catalase – in microbodies/peroxisomes of all
cells).
• Many enzymes located in membranes, such as
the ones on the inner mitochondrial membrane
(cellular respiration) and the chloroplast
membranes (photosynthesis). Digestive
enzymes embedded in the membranes of cells
of the small intestine (brush border).
Use of enzymes in biotechnology:
Example 1Lactase + Lactose
Intolerance
•
•
•
•
10% of Americans
10% of Africa’s Tutsi tribe
50% of Spanish and French people
99% of Chinese people
Lactose Intolerance
• Lactase gene is switched off after weaning.
• Stone-age ancestors of European dairy-lovers
couldn’t digest milk either.
Looked at 7,000 year old fossils of ancient
Europeans and could not find in their DNA the
lactose tolerance mutation. Seemed to have
evolved lactose tolerance around 5,000 years
ago.
Mutation arose independently in Africa around
7,000 years ago…very high frequency in
Tanzanian Hadza population
Lactase
• Lactose is disaccharide
found in milk.
• Converted by enzyme
lactase to glucose and
galactose.
• Lactase can be obtained
from the yeast,
Kluveromyces lactis.
• Used to help people who
are lactose intolerant.
• Used to break down
lactose in milk shakes
and fruit yoghurt so that
less sugar needs to be
used (glucose and
galactose are sweeter
than lactose).
• Used in ice cream to
create smoother texture
(lactose crystallizes)
• Can be used in making of
fermented cottage
cheese and yoghurt by
bacteria…faster.
Lactase can be used in 2 ways
during food processing.
• 1. Added to milk. Final product contains
the enzyme.
• 2. Immobilized on a surface or in beads of
a porous material. Milk is allowed to flow
past immobilized lactase. Avoids
contamination of the product with lactase.
Examples of the use of proteins in biotechnology: The use
of organisms or parts of organisms to produce things or to
carry out useful processes.
• Pectinase: obtained from
fungus, Aspergillus niger
• Breaks down the complex
polysaccharide, pectin,
found in the cell walls of
plants.
• Used during the crushing
of fruit to make juice more
fluid and easy to
separate. (Prevents
pectin from forming
cross-links and trapping
juice)
• Increases juice volume
and less cloudy.
• Protease: obtained from a
bacteria, Bacillus licheniformis,
that is adapted to grow in
alkaline conditions.
• Breaks down proteins into
soluble peptides and amino
acids.
• Used in detergents in laundry
washing powders to digest
protein.
• High pH optimum allows it to
remain active in alkalis.
• Allows lower temps to be used,
lower energy use, less
shrinkage or loss of colored
dyes.
Pectin
Review Guide Enzymes