15. Intracellular proteolysis

Question 1

Describe Proteolysis (3pts)

Answer 1

1. Is the reversal of protein synthesis
2. In proteolysis peptide bonds are cleaved during hydrolysis.
3. Catalysed by proteases

Question 2

Give 4 examples of Proteases(4pts)

Answer 2

1. Serine proteases- serine residues
2. Cystesine proteases- cystesine residues
3. Aspartly proteases- aspartly residue
4. Metalloproteases- form complexes with specific metal ions to be active.

Question 3

Name the two types of proteolysis? (2pts)

Answer 3

Endopeptidases and Exopeptidases

Question 4

Describe Endopepitases (1pts)

Answer 4

Cleaves proteins in the middle of the polypeptide chain

Question 5

Describe Exopeptidases

Answer 5

Remove one or two amino acids from the end of the polypeptide chain.
Aminopeptidases= work on the amino end
carboy peptidases= work on the carboy end.

Question 6

Describe the sequence specificity of Proteolysis?

Answer 6

Proteolysis is very specific. These proteins are recognised at a very specific site therefore a specific protein sequence is required.

Question 7

Describe protein activation in digestive enzymes by proteolysis? (5pts)

Answer 7

1. Chymotrypsin is inactive and is converted into alpha and pi- chymotrypsin by trypsin.
2. Trypsin cleaves the peptide bond in chymotrypsin.
3. This creates 2 peptides within n-chymotrypsin held together by a disulphide bond.
4. The activated n-chmotrypsin reacts with other n-chmotryspin molecules to cleave out 2 dipeptides which are Serine-14-Arginine-15 and Threonine-147-Aspagine-148.
5. This reaction yields the a-chymotrypsin. They are synthesised in the ER of the pancreas.

Question 8

Describe how insulin is activated by Proteolysis? (3pts)

Answer 8

• Insulin must be processed by proteolysis before it becomes active.

1. The initial protein Preproinsulin contains a signal sequence that is directed to the rough ER during synthesis.
2. The removal of the signal sequence produces pro-insulin.
3. Pro-insulin is cleaved via the c-peptide to produce the final mature insulin.

Question 9

Describe protein activation in the clotting cascade?

Answer 9

At each step of the cascade one serine protease cleaves another serine protease converting inactive zymogen to the active protease.

Question 10

Describe the royal disease?

Answer 10

Royal disease was caused by a mutation in the factor 9 gene. Queen Victoria got this mutation and passed it on to her offspring which causes no blood clots to form. This is on the x chromosome so usually only boys suffer from this gene. The mutation has been sequenced to work out what went wrong- in the factor 9 gene the A residue was changed to a G. The splicing occurred at the wrong place introducing two extra nucleotides to be added in the coding sequence.

Question 11

Describe Proteases in meat?

Answer 11

Proteases help break down tough fibres in meat making it easier to chew.

Question 12

Describe the introduction of HIV protease inhibitors?

Answer 12

Protease cleaves poly-protein precursors to form functional proteins. This led to the discovery of the HIVS crystal structure helping to develop drugs to treat AIDS.

Question 13

Describe Protein degradation?

Answer 13

Protein degradation is non-specific and is compartmentalised e.g occurs in lysosomes and the small intestine. Proteases formed in lysosomes operate at a low PH to avoid damage.

Question 14

Describe Protein degradation?

Answer 14

Protein degradation is non-specific and is compartmentalised e.g occurs in lysosomes and the small intestine. Proteases formed in lysosomes operate at a low PH to avoid damage.

Question 15

Describe Ubiqutin? (5pts)

Answer 15

1. Small protein
2. Highly conserved
3. 76 Amino acids
4. Molecular weight= 8.6 kDA
5. Ubiquitin attaches to target proteins that mark them for protein degradation.

Question 16

What is E1?

Answer 16

E1= Ubiquitin-activating enzyme

Question 17

What is E2?

Answer 17

E2= Ubiquitin-conjugating enzyme

Question 18

What is E3? (3pts)

Answer 18

E3= Ubiquitin Ligase

1. ensures specificity
2. recognises target proteins
3. Transports Ubiquitin from E2 to the target protein.

Question 19

Describe the 3-step pathway for Protein Ubiquitylation? (3pts)

Answer 19

1. The E1 enzyme binds to the Ubiquitin which involves a Cystesine molecule forming a thioester bond. Reaction requires ATP.
2. Ubiquitin is transferred from E1 to E2 to a Cytesine via a thioester bond.
3. Transfer of Ubuiqtin from E2 to a target protein via E3.

Question 20

How many molecules must be attached in order for the protein substrate to be marked for Proteolysis?

Answer 20

A minimum of 4 ubiquitin molecules must be attached in order for the protein substate to be marked for proteolysis.

Question 21

Describe the term half life? (3pts)

Answer 21

1. Half life is the amount of time required for half the original molecule to be degraded.
2. Different proteins have different half lives.
3. Some proteins are recognised and digested faster than others because they are more resistant and therefore act as better substrates.

Question 22

Describe the N-end rule pathway?

Answer 22

Many proteins are recognised by Ubiquitin ligases that attach to Ubiquitin molecules. The ligase recognise the N terminus of its target. Its affinity to the protein is determined by the amino acid at the end.