Lecture 4 Protein Modification and Protein Cleavage Flashcards
What is protein modification?
- The modification of selected residues in a protein
complex and not as a component of synthesis - Some modifications occur as the polypeptide chain is still being synthesised but not as part of the synthesis process
What are some examples of protein modifications?
- Acetylation
- Hydroxylation
- Glycosylation
- Phosphorylation
Describe Acetylation.
Acetylation refers to the addition of an acetyl group (CH3CO) to organic compounds
- Many proteins have their N-Terminal amino acid
modified by acetylation (making them more stable to degradation processes & extends a proteins lifetime) - The acetylation process involves the molecule Acetyl
Coenzyme A (Acetyl CoA)
-Acetylation is a feature ONLY of Eukaryotes NOT of
prokaryotes
- Between 59% and 90% of proteins in the cytoplasm are acetylated
- No proteins in chloroplasts are acetylated
Describe Hydroxylation and its effects.
Hydroxylation is the addition of an OH group to the
side chain of specific amino acids in a protein (proline & lysine)
- Hydroxyproline (Hyp) is an essential component of collagen (involved in the hydrogen bonding within the collagen fibre –> structural stability)
- There is a general repeating unit in the collagen sequence (Gly = Xaa = 4-Hyp)
- Between 15% and 30% of collagen is hydroxyproline
- 4-Hyp is formed by the enzyme prolyl hydroxylase, which requires ascorbic acid (Vitamin C) to function
- Vitamin C deficiency leads to a disease known as Scurvy (poor wound healing, teeth falling out, and in severe cases, death)
What is Glycosylation?
Glycosylation is the attachment of sugar molecules to
specific amino acids in a polypeptide chain
- ONLY in Eukaryotes NOT of Prokaryotes
- Two principal forms of glycosylation exist:
= N-Glycosylation
= O-Glycosylation
Describe N-Glycosylation.
- Sugars are attached to the Nitrogen in the side chain of the amino acid Asparagine (= Asn = N)
- The ‘N’ in N-Glycosylation refers to the Nitrogen
involved in the side chain linkage - These can be attached to proteins
- this aa sequence pattern must be present for N-Glycosylation to occur = N - X - S or N - X - T
- Where X is any amino acid except Proline
- Some possible N-Glycosylation sites are never used
- Sites which are used are not always glycosylated
Describe O-Glycosylation.
- Sugars are attached to the Oxygen in the side chain of the amino acids Serine or Threonine
- In O-Glycosylation there is no characteristic sequence pattern
- Proteins with added sugar molecules are more soluble
- The orientation of proteins in a membrane can be fixed by their attached sugar molecules
Describe Phosphorylation and it’s features.
Phosphorylation is the attachment of a phosphoryl group to the side chains of specific amino acids in a protein
- most common form of reversible protein modification
- Phosphorylation occurs on the side chain Oxygen atoms of Threonine, Serine, Tyrosine
- The phosphoryl group is attached to the amino acid by a member of an enzyme family, the Protein kinase (spec, tyrosine kinase)
- It is removed from the amino acid by a member of an
enzyme family the Protein Phosphatases - Phosphorylation is a central feature of enzyme regulation
- Some members of the Protein Kinase family are highly selective and recognise characteristic sequences
- Example: Protein Kinase A recognises: Arg - Arg - Xaa - Ser - Zaa or Arg - Arg - Xaa - Thr - Zaa
- Where Xaa is a small residue and Zaa is a large
hydrophobic residue
Define Protein Cleavage.
Process of breaking the peptide bonds between amino acids in proteins (by enzyme protease)
What examples of protease exist?
Three examples of different types of protease are:
- Carboxypeptidase A
- Chymotrypsin
- HIV Protease
How does Carboxypeptidase A function?
- Carboxypeptidase A is a member of a large enzyme family the Metalloproteases
- This family has a metal ion at the enzyme active site
- In carboxypeptidase A this is zinc (zinc (II))
Function:
- Carboxypeptidase A cleaves off the last C-Terminal
residue from a polypeptide chain
- The enzyme works best when this is either an
aromatic or a bulky aliphatic residue
How does Chymotrypsin function?
- Chymotrypsin is a member of the Serine Protease family
- This family has an important active site serine residue
Function:
- Chymotrypsin cleaves peptide bonds on the carboxyl side of peptide chains or large hydrophobic residues
How does HIV protease function?
- HIV Protease is a member of the Aspartic Protease family
- Aspartate residues are central to the active site
Function:
- The first role of HIV Protease is to cleave itself out of a large chain protein formed from the genetic material of the virus
- Then HIV protease cleaves out the remaining proteins of the virus
- The function of HIV Protease is critical to viral
replication
- The protease has become an important target for drug development
- The HIV Protease Inhibitors have been produced as
a result
Why do pre-proteases and proteases exist?
- Many polypeptides are synthesised as longer chains than their native chain length and require cleavage
Reasons:
- The added sequence acts to direct a protein to specific compartments within a cell
- The longer chain assists in folding the protein correctly
- The longer chain renders the protein inactive
+ Example: Chymotrypsin is formed as an inactive
Prochymotrypsin and is only activated when cleaved to
the native chain length
- Needed for the correct transportation, for inactivation, and for the correct folding of proteins
