This lecture is about zymogens. This is about how proteins digest other proteins.
- Proteolysis - the breakdown of peptide bonds that link amino acids to form proteins - is used to:
- Control protein activity by activating or deactivating some proteins (lysis of some proteins can make them active - e.g. you produce a protein in an inactive form, and then activate it by cleavage)
- Release the amino acids from proteins for use as building blocks for new proteins
Activation by specific proteolytic cleavage
- Not all proteins are active when produced and folded into their 3-D conformation (e.g. insulin - this has slowed down the development of bacterial insulin. Bacteria can't cleave/fold the full-length insulin for use in humans)
- They require activation by specific proteolytic cleavage
- The inactive precursor is called a zymogen or proenzyme
- Energy (ATP) is not required for this cleavage
- Can occur outside cells
- Occurs once only in the life of the protein
- Digestive enzymes
- Blood clotting
- If activated at site of production, it would cause unnecessary clotting (need clotting at the site of trauma)
- Some hormones (e.g. insulin)
- Some structural proteins (e.g. collagen)
- Developmental processes
- Apoptosis (programmed cell death) - caspases
- Ingested proteins are not absorbed intact
- They are ultimately broken down into amino acids and di- and tripeptides for absorption and transport
- Begins in the stomach where the acidic environment denatures the proteins
- Denatured proteins are digested further by pepsin - a protease that is maximally active at pH 2
- Continues in the lumen of the intestine using proteolytic enzymes from the pancreas
- Aminopeptidases in the plasma membrane of the intestinal cells complete the digestion
- Overview: Protein, starts to be broken down in the stomach, gets broken down further in the intestinal cell, then transported into the blood.
- Some proteins are cleaved into mono, di and tripeptides. Others are cleaved into oligopeptides. The oligopeptides are cleaved down to di and tripeptides by enzymes on the cell surface as they are transported into the cell. These can then be transported into the blood.
It is essential that the proteases are not active until:
- in the correct location (not where they are synthesised)
- there are target proteins to digest.
Pepsinogen is produced in the stomach, the others are produced in the pancreas
- The pancreas is one of the most active organs for synthesis and secretion of proteins in the body.
- Zymogens are synthesized in the acinar cells of the pancreas
The zymogen granules are the pink inclusions in the pancreatic exocrine cells. The pancreas produces very large amounts of these zymogens, and they need to be removed and activated elsewhere (duodenum) for digestion.
Secretion of zymogens
- Zymogen granules accumulate at the apex of the acinar cells in the pancreas.
- Hormonal signaling or nerve impulse causes their release into a duct leading to the duodenum.
Produced in ribosomes on rER, transported to Golgi apparatus, and the vesicles are released from the cell upon nervous or other activation indicating a protein bolus has been ingested.
- 245 amino acids
- Devoid of enzyme activity
- Proteolysis leads to activation
- Active enzyme is chymotrypsin
Activation of chymo-trypsinogen
- Cleavage of a single peptide bond is sufficient to activate the enzyme.
- After the first step, the enzyme has partial activity and can carry out autolysis, to split the enzyme into 3 segments at the end
- α-chymotrypsin is the stable form of the enzyme.
- It has 3 polypeptide chains linked by disulphide bonds.
- The cleavage leaving behind disulphide bonds produces highly flexible locations where the cleavage occurred, allowing the enzyme to fold into the correct conformation
Proteolysis of chymotrypsinogen
Proteolysis of chymotrypsinogen allows movement of amino acids into configurations that are essential for the structure of the active enzyme i.e. formation of the substrate binding site and the active site e.g the movement of Ile 16 close to Asp 194. Cutting and release of the enzyme allows Ile 16 to move close to Asp 194, which is essential to the function of the enzyme.
Specificity of chymotrypsin
- This is the reason there are many digestive enzyme. One may cleave one part of the peptide, and then another enzyme will cleave the product of this.
- E.g. chymotrypsin cleaves peptides that occur after large hydrophobic groups
- Therefore it won't cleave all peptides
- Then we need other proteolytic enzymes
Other proteolytic enzymes
- The digestive proteins trypsin and elastase are homologues (things of similar structure) of chymotrypsin
- Their overall structures are nearly identical
- Very different substrate specificities (and their substrates are other proteins)
- How does this work?
Structural similarity of trypsin and chymotrypsin
- Enzymes cleave the peptide backbone after aa with specific types of side chains
- Chymotrypsin cleaves after hydrophobic peptides
- Trypsin cleaves after positively charged peptides
- Elastase cleaves after small side chains (e.g. H, CH3)
The importance of the active site
- Changing amino acids in the active sites of these three enzymes changes their specificity
- The differences in substrate specificity are produced by changes in a few amino acids at the substrate binding site
- e.g. Chymotrypsin has no charged amino acids in its substrate binding site (active site) - therefore more adapted to cleave hydrophobic peptides
- Similarly, trypsin has Asp in its binding site, which is negatively charged, so it prefers to bind and cleave positively charged amino acids
- Elastase has two bulky groups (Val 190 and Val 216), leaving only a small space in the active site to bind substrate, therefore it only cleaves after small groups.
- Their similarity in structure is in the mechanism of the catalytic part of the enzyme that actually cleaves a peptide bond (this is the same process for all of these enzymes)
￼Digestion of proteins in the gut
- Each proteolytic enzyme has a different substrate specificity
- So, concurrent activation of several proteolytic enzymes is required
- Achieved by trypsin acting as the common activator of all pancreatic zymogens (including itself)
- Trypsin itself is produced as a zymogen (trypsinogen) - it cleaves all of the active sites of the enzymes to activate them
- The cells lining the duodenum secrete enteropeptidase which cleaves and activates trypsinogen
Digestive enzyme activation
- As the enzymes move into the lumen, enteropeptidase is active, which activates trypsinogen into trypsin (note also that trypsin can cleave trypsinogen, which then produces more trypsin (autolytic))
- Trypsin then is then able to activate all the other zymogens (it's very precise but it's irreversible)
- NB: carboxypeptidase cleaves the amino acids from the carboxy terminal of the polypeptide
Note: the formation of trypsin by enteropeptidase is the master activation step
￼Regulation of trypsin activity
- The conversion of the inactive trypsinogen to the active trypsin is a very precise mechanism, but it is irreversible (goes around cleaving and activating zymogens without thinking)
- The pancreas also synthesises a trypsin inhibitory protein - this stops the trypsin from going psycho in the pancreas
Pancreatic Trypsin Inhibitor (PTI)
- Part of the PTI polypeptide chain is recognised as a substrate by trypsin and binds tightly at the active site
- But the PTI is a very poor substrate and is only cleaved very slowly
- Competitive inhibitor
Why synthesise PTI?
- The PTI acts as an off-switch for any trypsin formed by accidental trypsinogen activation.
- Accidental activation of trypsin is particularly dangerous because of its position at the start of the proteolytic cascade of digestion enzymes.
- If trypsin is activated in the pancreas or the pancreatic ducts it will lead to pancreatitis or tissue necrosis.
- Another protease inhibitor
- Binds irreversibly to elastase, a secretory product of neutrophils (which phagocytose things)
- Its binding protects tissues from digestion
- Genetic disorders leading to a deficiency in the enzyme causes emphysema
- Excess elastase destroys alveolar walls in the lung by digesting elastic fibres and connective tissue proteins
Why does cigarette smoke cause emphysema?
- Cigarette smoke oxidises a methionine in the α1-antitrypsin.
- This prevents the inhibitor from binding elastase.
- The methioinine acts as a bait to selectively trap the elastase.
- The addition of a single O is sufficient to prevent this.
￼Cellular protein turnover
- Proteins within cells are also degraded
- This provides a mechanism for regulating cellular behaviour and metabolism (e.g. in cell cycle, when we move from S to G2, we need to degrade all the S proteins to signal the end of that phase)
- Cells also detect and remove damaged or defective proteins
- The half-lives of intracellular proteins varies from minutes to weeks (Hb) to years (crystallin)
- Proteases cleaving zymogens that become proteases that digest other proteins