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 modulatingthe levels of small signalling molecules. The binding of thesemolecules causes conformational changes in the proteinwhich affect its activity.

Multiple forms of Enzymes:Different tissues or developmental stages sometimes have specificversions of a given enzyme which have distinct properties althoughthey may have the same basic activity.

Reversible Covalent Modification:The activity of many proteins is controlled by attachment of smallchemical groups. The most common such modification isphosphorylation- attachment of a phosphate group.

Proteolytic Activation:Some enzymes are synthesized in an inactive form and must beactivated by cleavage of the inactive form.

Some enzymes are synthesized in an initially inactive (but folded) form which is converted to an active form by specific proteolyticcleavage.

These initial forms are called zymogens or proenzymes.

This method of regulation does not require an energy sourceunlike phosphorylation which requires ATP.

Therefore extracellular enzymes may be activated by this process.

Proteolysis is irreversible- once activated, the molecule remainsin the activated state.

Zymogens

Examples of Proteolytic Activation

Digestive Enzymes: The primary enzymes that function in breakingdown proteins and peptides during digestion are synthesized as zymogensin the stomach and pancreas.

Blood Clotting: Rapid response to injury is possible by activating acascade of zymogens.

Hormones: Some hormones, e.g. insulin, are synthesized as precursorswhich must be activated by proteolysis.

Collagen: The major component of skin and bone, collagen is derivedfrom its precursor procollagen by specific proteolysis.

Developmental Processes: The structural protein collagen must be brokendown in certain tissues at particular stages during normal development.The protease responsible for this process, collagenase, is activated at theprecise 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 developmentand also functions to eliminate cells that are somehow damaged, eg infectedwith pathogens or containing DNA too damaged to repair. This processis mediated by proteolytic enzymes called caspases, which are initiallysynthesized as inactive procaspases and can be activated by proteolysisin response to a variety of signals.

Digestive Zymogens

The pancreas is a major producer ofdigestive 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 zymogensare all activated by a single enzyme, trypsin.

Trypsin itself is activated by enteropeptidase, which is secreted bycells lining the digestive tract. In turn trypsin activates the other zymogens.

Activation of Chymotrypsin

Chymotrypsin is initially synthesized as the inactive precursorchymotrypsinogen. Initial cleavage by trypsin yields -chymotrypsin,which is further processed by chymotrypsin itself to yield -chymotrypsin,the final active form.

Structural Basis of Chymotrypsin Activation

Comparison of the structures of chymotrypsin and chymotrypsinogenrevealed that the inactive and active forms are very similar overall but that small, local rearrangements exist that explain the difference inactivity.

The break at Ile 16 creates a newpositive amino terminus which formsa buried ionic interaction with Asp 194.

Subsequent rearrangements causethe formation of a hydrophobic cavityimportant for substrate specificity,and also formation of the oxyanionhole which is required for thethe catalytic activity of theactivated enzyme.

Inhibition of Trypsin

The accidental activation of a few trypsin molecules inside the acinarcells could be disastrous. A small amount of active trypsin could activateall 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 cannotdissociate the complex.

The tight binding is partly conferredby a Lys side-chain which bindsin a negatively charged pocket ontrypsin.

PTI is eventually cleaved by trypsinbut only extremely slowly (overmonths) and the combination of tightbinding and slow hydrolysis makesit 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 ofconnective tissue proteins.

White blood cells secrete elastase, which is a protease that is capable ofdegrading elastic fibers.

Normally this is prevented by a protein in blood plasma called1-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 issecreted 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 1-antiproteinase- the smoke oxidizes a particular methionine residue on 1-antiproteinase:

This residue is an essential part of the recognition interface between elastase allowing it to bind 1-antiproteinase.

When this methionine is oxidized, the binding is disrupted, the 1-antiproteinase can no longer inhibit elastase, and elastasedegrades the elastic fibers in the lungs, leading to emphysema.

Smoking is particularly dangerous for persons with a geneticdefect in the inhibitor.

Methionine Methioninesulfoxide

Activation Cascades

Rapid response to a stimulus is possible through a cascade of enzymeactivations. A cascade consists of a series of several steps each of whichhas a multiplicative effect on subsequent steps.

Step 1: A signalling molecule activates 1 moleculeof enzyme 1.

Step 2: Enzyme 1 activates 100 moleculesof enzyme 2. (100-fold amplification)

Step 3: Each activated molecule of enzyme 2 activates 100molecules of enzyme 3. (104-fold activation)

Step 4: Each activated molecule of enzyme 3 activates 100molecules of enzyme 4. (106-fold activation)

Cascades can produce an enormous and extremely rapid response. Anexample of such a process occurs in blood clotting.

Blood Clotting: A Cascade of Zymogen ActivationsThe clotting of blood after injury must be rapid to avoid blood loss. Therapidity with which this is accomplished is due to a cascade of activationof blood clotting factors. Small amounts of the initial clotting factorsamplify the response and result in the rapid formation of clots.

Clotting factors are referred to byRoman numerals.

These were named in the order thatthey were discovered, not for theorder in which they act.

The inactive zymogen form isdenoted by the Roman numeral,(e.g. Factor X) and the activatedform is indicated by adding thesuffix “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 releaseof Tissue factor, a lipoprotein.

Both pathways converge in the final steps, in which the protease thrombinis activated and releases theclot-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.

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 A, B, and .

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 FibrinThe new termini of the chain created when the A peptides are cleavedoff by thrombin interact with binding sites on the subunit.The fibers are further stabilized by amide crosslinks betweenfibrin monomer side-chains.

Bindingsite

Fibrin arrayand electronmicrograph

Transglutaminase

Cessation of Clot Formation

The cascade of activations during clot formation must be carefullyregulated 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 factorsthrough dilution, removal from the bloodstream, and by proteolysis. Specificinhibitors to individual clotting factors (serpins) exist which also attenuatethe 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.

Blood vessel in heartblocked by clot

Blood flow restored:Blockage removed afterTPA was administered

Removal of Clots

When no longer required clots are removed by proteolysis of fibrinby the protease plasmin. Plasmin is itself originally produced as aninactive precursor, plasminogen, which is released through the actionof tissue-type plasminogen activator (TPA). TPA is given to someheart attack victims to restore circulation through blocked blood vessels.

Summary:

Zymogens are inactive protein precursors which must be converted to theiractive forms by specific proteolytic cleavage events.

A variety of digestive enzymes are synthesized as zymogens in thepancreas. They are activated by proteolysis, and further control of theiractivities is achieved through the action of specific inhibitor proteins.

A cascade of zymogen activations resulting in the controlled creationof fibrin aggregates is the molecular basis of blood clotting.

Key Concepts:Zymogens

Control of activationRoles of inhibitor proteins (Serpins)Emphysema

Activation CascadesMechanism of blood clottingHemophilia