IT - Proteases Flashcards
Where are proteases found?
All organisms (viruses, prokaryotes, eukaryotes)
What are some functions of proteases? (2)
- Digestion of food
- Regulated cascades (blood clotting, apoptosis)
What are the main types of protease catalytic mechanisms? (4)
- Serine triad protease
- Cysteine dyad protease
- Metalloprotease
- Aspartate protease
Are Trypsin and subtilisin structurally similar?
No, they are serine triad proteases with different structures
Why are triad enzymes a strong case for convergent evolution? (2)
- There is no sequence homology between triad enzymes
- There is no structural homology between triad enzymes
What are the three amino acids in the catalytic triad?
A. Cysteine/Serine (nucleophile)
B. Histidine (base)
C. Aspartate/Glutamate (acid)
How is an oxyanion formed?
The proton attached to cysteine/serine is removed (stronger nucleophile formed)
- Forms acyl intermediate (Oxyanion)
What are features of Oxyanions? (4)
- unstable
- short-lived
- will revert to starting materials
- need for stabilisation (=>lower the pKa of the oxyanion!)
Protease Mechanism - Peptide Bond Cleavage (6)
- Cleaved proton transfers to N-H on the substrate.
- C-N bond is cleaved.
- N-H2-R group diffuses out.
- Carbonyl group reforms.
- Water molecule acts as a nucleophile.
- The nucleophile takes the hydrogen back from the base (histidine) to restore the catalytic triad.
What is the role of the oxyanion hole? (2)
- a hole to stabilise the transition state
- acts by lowering the pKa of the oxyanion
What are the four key features of a protease active site?
- Catalytic triad
- Substrate binding cleft
- Oxyanion hole
- Specificity pocket
How does substrate specificity differ between Trypsin and Subtilisin?
- Trypsin cleaves after positively charged residues (Arg, Lys)
- Subtilisin cleaves after hydrophobic residues (Phe, Trp, Tyr)
Give an example of a high-stability, low-specificity protease.
Subtilisin (serine protease)
What is an example of a protease that cleaves signal peptides?
Signal peptidase
Three examples:
- ER-targeting
- Bacterial leader-peptidases
- Mitochondrial signal peptidases of the inner mitochondrial membrane
What type of protease is involved in the blood clotting cascade?
Serine protease
Examples:
- Thrombin and factors VII, IX, X, XI
Factors V and VIII are coagulation factors (no proteolytic activity)
What are 4 key facts about Caspases?
- Cysteine proteases that cleave proteins at the carboxy-terminal side of Asp residues
- Involved in controlled cell death/apoptosis
- Involved in differentiation, proliferation, immune system regulation
- Neurodegenerative disease: Chorea Huntington
Initiatior caspases vs Executionar caspases
Initiator caspase (8,9)
inactive monomers associate
Executioner caspase (3,6,7)
inactive dimers rearrange
What are cysteine proteases?
Catalytic dyad with cysteine as nucleophile
No acid required
E.g
- Papain
- Bromelain
- Caspase
