IT - Proteases

Question 1

Where are proteases found?

Answer 1

All organisms (viruses, prokaryotes, eukaryotes)

Question 2

What are some functions of proteases? (2)

Answer 2

1. Digestion of food
2. Regulated cascades (blood clotting, apoptosis)

Question 3

What are the main types of protease catalytic mechanisms? (4)

Answer 3

1. Serine triad protease
2. Cysteine dyad protease
3. Metalloprotease
4. Aspartate protease

Question 4

Are Trypsin and subtilisin structurally similar?

Answer 4

No, they are serine triad proteases with different structures

Question 5

Why are triad enzymes a strong case for convergent evolution? (2)

Answer 5

• There is no sequence homology between triad enzymes
• There is no structural homology between triad enzymes

Question 6

What are the three amino acids in the catalytic triad?

Answer 6

A. Cysteine/Serine (nucleophile)
B. Histidine (base)
C. Aspartate/Glutamate (acid)

Question 7

How is an oxyanion formed?

Answer 7

The proton attached to cysteine/serine is removed (stronger nucleophile formed)

• Forms acyl intermediate (Oxyanion)

Question 8

What are features of Oxyanions? (4)

Answer 8

• unstable
• short-lived
• will revert to starting materials
• need for stabilisation (=>lower the pKa of the oxyanion!)

Question 9

Protease Mechanism - Peptide Bond Cleavage (6)

Answer 9

1. Cleaved proton transfers to N-H on the substrate.
2. C-N bond is cleaved.
3. N-H2-R group diffuses out.
4. Carbonyl group reforms.
5. Water molecule acts as a nucleophile.
6. The nucleophile takes the hydrogen back from the base (histidine) to restore the catalytic triad.

Question 10

What is the role of the oxyanion hole? (2)

Answer 10

• a hole to stabilise the transition state
• acts by lowering the pKa of the oxyanion

Question 11

What are the four key features of a protease active site?

Answer 11

• Catalytic triad
• Substrate binding cleft
• Oxyanion hole
• Specificity pocket

Question 12

How does substrate specificity differ between Trypsin and Subtilisin?

Answer 12

• Trypsin cleaves after positively charged residues (Arg, Lys)
• Subtilisin cleaves after hydrophobic residues (Phe, Trp, Tyr)

Question 13

Give an example of a high-stability, low-specificity protease.

Answer 13

Subtilisin (serine protease)

Question 14

What is an example of a protease that cleaves signal peptides?

Answer 14

Signal peptidase

Three examples:

• ER-targeting
• Bacterial leader-peptidases
• Mitochondrial signal peptidases of the inner mitochondrial membrane

Question 15

What type of protease is involved in the blood clotting cascade?

Answer 15

Serine protease

Examples:

• Thrombin and factors VII, IX, X, XI

Factors V and VIII are coagulation factors (no proteolytic activity)

Question 16

What are 4 key facts about Caspases?

Answer 16

• 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

Question 17

Initiatior caspases vs Executionar caspases

Answer 17

Initiator caspase (8,9)
inactive monomers associate

Executioner caspase (3,6,7)
inactive dimers rearrange

Question 18

What are cysteine proteases?

Answer 18

Catalytic dyad with cysteine as nucleophile

No acid required

E.g

• Papain
• Bromelain
• Caspase