Lecture 17: Regulation of Proteins 4: Proteolytic Activation

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Lecture 17Regulation of Proteins 4Proteolytic
Activation

• Examples
• Activation of Digestive Enzymes
• Blood Clotting

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Biological Processes are Carefully Regulated
Allosteric Control The activity of some
proteins can be controlled by modulating the
levels of small signalling molecules. The binding
of these molecules causes conformational changes
in the protein which affect its
activity. Multiple forms of Enzymes Different
tissues or developmental stages sometimes have
specific versions of a given enzyme which have
distinct properties although they may have the
same basic activity. Reversible Covalent
Modification The activity of many proteins is
controlled by attachment of small chemical
groups. The most common such modification
is phosphorylation- attachment of a phosphate
group. Proteolytic Activation Some enzymes are
synthesized in an inactive form and must
be activated by cleavage of the inactive form.
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Zymogens
Some enzymes are synthesized in an initially
inactive (but folded) form which is converted to
an active form by specific proteolytic cleavage.
These initial forms are called zymogens or
proenzymes. This method of regulation does not
require an energy source unlike phosphorylation
which requires ATP. Therefore extracellular
enzymes may be activated by this
process. Proteolysis is irreversible- once
activated, the molecule remains in the activated
state.
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Examples of Proteolytic Activation
Digestive Enzymes The primary enzymes that
function in breaking down proteins and peptides
during digestion are synthesized as zymogens in
the stomach and pancreas.
Blood Clotting Rapid response to injury is
possible by activating a cascade of zymogens.
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Hormones Some hormones, e.g. insulin, are
synthesized as precursors which must be activated
by proteolysis. Collagen The major component
of skin and bone, collagen is derived from its
precursor procollagen by specific
proteolysis. Developmental Processes The
structural protein collagen must be broken down
in certain tissues at particular stages during
normal development. The protease responsible for
this process, collagenase, is activated at
the precise time needed by specific
proteolysis. Apoptosis Cells have an intrinsic
ability to self-destruct. This
process, programmed cell death or apoptosis, is
required during normal development and also
functions to eliminate cells that are somehow
damaged, eg infected with pathogens or containing
DNA too damaged to repair. This process is
mediated by proteolytic enzymes called caspases,
which are initially synthesized as inactive
procaspases and can be activated by
proteolysis in response to a variety of signals.
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Digestive Zymogens
The pancreas is a major producer of digestive
enzymes. Acinar cells in the pancreas produce a
variety of zymogens which are stored in
membrane-bounded granules. These zymogen
granules fuse with the cell membrane in response
to signals from hormones or nerve
impulses, releasing their contents into ducts
leading to the digestive tract. The zymogens
include trypsinogen, chymotrypsinogen,
proelastase, and procarboxypeptidase.
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Activation of Digestive Zymogens
The different digestive proteases have different
substrate specificities, enabling the breakdown
of a wide variety of peptides. The zymogens are
all activated by a single enzyme, trypsin.
Trypsin itself is activated by enteropeptidase,
which is secreted by cells lining the digestive
tract. In turn trypsin activates the other
zymogens.
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Activation of Chymotrypsin
Chymotrypsin is initially synthesized as the
inactive precursor chymotrypsinogen. Initial
cleavage by trypsin yields p-chymotrypsin, which
is further processed by chymotrypsin itself to
yield a-chymotrypsin, the final active form.
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Structural Basis of Chymotrypsin Activation
Comparison of the structures of chymotrypsin and
chymotrypsinogen revealed that the inactive and
active forms are very similar overall but that
small, local rearrangements exist that explain
the difference in activity.
The break at Ile 16 creates a new positive amino
terminus which forms a buried ionic interaction
with Asp 194. Subsequent rearrangements
cause the formation of a hydrophobic
cavity important for substrate specificity, and
also formation of the oxyanion hole which is
required for the the catalytic activity of
the activated enzyme.
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Inhibition of Trypsin
The accidental activation of a few trypsin
molecules inside the acinar cells could be
disastrous. A small amount of active trypsin
could activate all the zymogens which would lead
to digestion of all the proteins in the cell. To
guard against this possibility, the acinar cells
contains a small (6 kD )protein that inhibits
trypsin- pancreatic trypsin inhibitor or PTI.
PTI binds extremely tightly to trypsin- even 8M
urea or 6M HCl cannot dissociate the
complex. The tight binding is partly
conferred by a Lys side-chain which binds in a
negatively charged pocket on trypsin. PTI is
eventually cleaved by trypsin but only extremely
slowly (over months) and the combination of
tight binding and slow hydrolysis makes it a very
effective inhibitor.
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Emphysema
Emphysema can result from a defect in a similar
type of inhibitor. Emphysema is a result of
loss of elasticity in the alveolar walls of the
lungs, reducing the volume in the lungs available
for exchange of O2 and CO2. This loss of
elasticity is caused by damage to elastic fibers,
composed of connective tissue proteins. White
blood cells secrete elastase, which is a protease
that is capable of degrading elastic fibers.
Normally this is prevented by a protein in
blood plasma called a1-antiproteinase that binds
to and inhibits the secreted elastase, protecting
your lungs from damage. People with inherited
disorders in this inhibitor or its production (it
is secreted by the liver) are at much higher risk
for developing emphysema. There is a family of
such inhibitors, called serpins, which is short
for Serine Protease Inhibitors.
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Connection between Smoking and Emphysema
Tobacco smoke contributes to emphysema by
damaging a1-antiproteinase- the smoke oxidizes a
particular methionine residue on
a1-antiproteinase
Methionine sulfoxide
Methionine
This residue is an essential part of the
recognition interface between elastase allowing
it to bind a1-antiproteinase. When this
methionine is oxidized, the binding is disrupted,
the a1-antiproteinase can no longer inhibit
elastase, and elastase degrades the elastic
fibers in the lungs, leading to
emphysema. Smoking is particularly dangerous for
persons with a genetic defect in the inhibitor.
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Activation Cascades
Rapid response to a stimulus is possible through
a cascade of enzyme activations. A cascade
consists of a series of several steps each of
which has a multiplicative effect on subsequent
steps.
Step 1 A signalling molecule activates 1
molecule of enzyme 1. Step 2 Enzyme 1
activates 100 molecules of enzyme 2. (100-fold
amplification) Step 3 Each activated molecule
of enzyme 2 activates 100 molecules of enzyme 3.
(104-fold activation) Step 4 Each activated
molecule of enzyme 3 activates 100 molecules of
enzyme 4. (106-fold activation)
Cascades can produce an enormous and extremely
rapid response. An example of such a process
occurs in blood clotting.
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Blood Clotting A Cascade of Zymogen Activations
The clotting of blood after injury must be rapid
to avoid blood loss. The rapidity with which this
is accomplished is due to a cascade of
activation of blood clotting factors. Small
amounts of the initial clotting factors amplify
the response and result in the rapid formation of
clots.
Clotting factors are referred to by Roman
numerals. These were named in the order
that they were discovered, not for the order in
which they act. The inactive zymogen form
is denoted by the Roman numeral, (e.g. Factor X)
and the activated form is indicated by adding
the suffix a. (e.g. Factor Xa)
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Two Pathways of Blood Clotting
The blood-clotting cascade can be activated in
two different ways. The intrinsic pathway is
initiated by exposure of abnormal surfaces of
ruptured blood vessels. The extrinsic pathway
is initiated by trauma, resulting in the by the
release of Tissue factor, a lipoprotein. Both
pathways converge in the final steps, in which
the protease thrombin is activated and releases
the clot-forming protein fibrin from its
precursor fibrinogen.
Hemophilia results from the loss of Factor VIIIa,
which partially or wholly blocks the intrinsic
pathway. The resulting inability to form clots
can make even a small wound life-threatening.
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Final Steps in Clot Formation
Clots consist largely of ordered fibrous arrays
of the protein fibrin. Fibrin is cleaved from
its zymogen fibrinogen by the protease
thrombin. When released from fibrinogen, fibrin
rapidly polymerizes into ordered arrays. These
arrays are further stabilized by covalent
crosslinks between fibrin monomers.
Activation
Fibrin release
Crosslinking
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Fibrinogen and Fibrin
Fibrinogen constitutes 2-3 of blood plasma
protein. It exists as a complex of 3 subunits
Aa, Bb, and g. Small peptides A and B are
removed by thrombin to release fibrin, revealing
creating new termini which enable fibrin to
polymerize into fibers.
Fibrinogen
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Clot Formation by Fibrin
The new termini of the a chain created when the A
peptides are cleaved off by thrombin interact
with binding sites on the g subunit. The fibers
are further stabilized by amide crosslinks
between fibrin monomer side-chains.
Binding site
Fibrin array and electron micrograph
g
Transglutaminase
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Cessation of Clot Formation
The cascade of activations during clot formation
must be carefully regulated so that clots will
not continue to expand more than necessary, which
would block blood flow to healthy tissue
(thrombosis). Once initiated, the clotting
cascade is attenuated by loss of clotting
factors through dilution, removal from the
bloodstream, and by proteolysis.
Specific inhibitors to individual clotting
factors (serpins) exist which also attenuate the
cascade.
Protein C is a protease that degrades factors Va
and VIIIa. It is activated by thrombin. Once
the final steps of the cascade are reached, the
factors carrying out the prior steps are
deactivated.
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Removal of Clots
When no longer required clots are removed by
proteolysis of fibrin by the protease plasmin.
Plasmin is itself originally produced as
an inactive precursor, plasminogen, which is
released through the action of tissue-type
plasminogen activator (TPA). TPA is given to
some heart attack victims to restore circulation
through blocked blood vessels.
Blood flow restored Blockage removed after TPA
was administered
Blood vessel in heart blocked by clot
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Summary
Zymogens are inactive protein precursors which
must be converted to their active forms by
specific proteolytic cleavage events. A variety
of digestive enzymes are synthesized as zymogens
in the pancreas. They are activated by
proteolysis, and further control of
their activities is achieved through the action
of specific inhibitor proteins. A cascade of
zymogen activations resulting in the controlled
creation of fibrin aggregates is the molecular
basis of blood clotting. Key Concepts Zymogens
Control of activation Roles of inhibitor proteins
(Serpins) Emphysema Activation
Cascades Mechanism of blood clotting Hemophilia