Transcript Slide 1

Lecture 17:
Regulation of Proteins 4:
Proteolytic Activation
Examples
Activation of Digestive Enzymes
Blood Clotting
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.
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.
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.
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.
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.
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.
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.
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.
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 trypsineven 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.
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.
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
Methionine
sulfoxide
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.
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.
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)
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.
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 lifethreatening.
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.
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
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
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.
Fibrin array
and electron
micrograph
Binding
site
g
Transglutaminase
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.
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 vessel in heart
blocked by clot
Blood flow restored:
Blockage removed after
TPA was administered
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