Lecture Four: Protein Modification & Cleavage
[Based on Chapters 2, 9, 10 & 11 – Berg, Tymoczko &
Stryer]
(Figures in red are for the 7th Edition)
 Protein Modification Overview
 DEFINITION
 The modification of selected residues in a protein
____________________ 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
 Modifications covered
 __________
 Hydroxylation
 Glycosylation
 ______________
 ACETYLATION
 Many proteins have their N-Terminal amino acid
modified by acetylation
 The acetylation process involves the molecule Acetyl
Coenzyme A
 Acetyl Coenzyme A = Acetyl CoA
O

H3C  C  S  CoA + H2N  Protein 
O

H3C  C  HN  Protein + H  S  CoA
 Acetylation is a feature ONLY of Eukaryotes NOT of
___________
 Between 59% and 90% of proteins in the cytoplasm are
acetylated
 No proteins in either ___________ or chloroplasts are
acetylated
 Conclusion: Acetylation does not take place in
these organelles
 Proteins with N-Terminal acetylation are more stable to
degradation processes than proteins without N-Terminal
acetylation
 Acetylation extends a proteins lifetime from between
seconds and hours up to days
 HYDROXYLATION
 Hydroxylation is the addition of an OH group to the
side chain of specific amino acids in a protein
 _______
 Lysine
 Hydroxyproline is often abbreviated to Hyp
 Figure 2-67, page 57 (2-67, page 54)
 Hydroxyproline is an essential component of collagen
 It is involved in the hydrogen bonding within the
collagen fibre
 This is a key feature of structural stability
 There is a general repeating unit in the collagen sequence
 Gly  Xaa  Yaa
 Xaa and Yaa are two other amino acids
 Between 15% and 30% of collagen is hydroxyproline
 4-Hydroxyproline is found in the Yaa position
 4-Hyp is formed by the enzyme prolyl hydroxylase
 This enzyme requires ascorbic acid to function
 Ascorbic acid = ___________
 Vitamin C deficiency leads to a disease known as Scurvy
 Scurvy symptoms are poor wound healing, teeth
falling out, and in severe cases, death
 GLYCOSYLATION
 Glycosylation is the attachment of sugar molecules to
specific amino acids in a polypeptide chain
 Glycosylation is a feature ONLY of Eukaryotes NOT of
Prokaryotes
 Two principal forms of glycosylation exist
 N-Glycosylation
 O-Glycosylation
 N-Glycosylation
 Figure 11-19 & Figure 11-20, page 316 (11-15 & 11-16, p. 340)
 Sugars are attached to the Nitrogen in the side chain of the
amino acid _____________ (= Asn = N)
 The ‘N’ in N-Glycosylation refers to the Nitrogen
involved in the side chain linkage
 Figure 11-21, page 317 (11-17, p 341)
 These can be attached to proteins
 A characteristic sequence pattern must be present for
N-Glycosylation to occur
 Asn  Xaa  Ser
or
Asn  Xaa  Thr
 In one letter form
 NXS
or
NXT
 Where Xaa is any amino acid except Proline
 Some possible N-Glycosylation sites are never used
 Sites which are used are not always glycosylated
 O-Glycosylation
 Figure 11-19, page 316 (11-15, p.340)
 Sugars are attached to the Oxygen in the side chain of the
amino acids Serine (= Ser = S) or ___________
(= ____ = T)
 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
 PHOSPHORYLATION
 Phosphorylation is the attachment of a phosphoryl
group to the side chains of specific amino acids in a
protein
 This is the most common and ubiquitous form of
reversible protein modification
 Phosphorylation occurs on the side chain Oxygen atoms of
 Threonine
 Serine
 _________
 [ Figures, page 285 ] [Figures, pages 309, 310]
 The phosphoryl group is attached to the amino acid by a
member of an enzyme family, the Protein ________
 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
 PROTEIN CLEAVAGE
 Amino acids are cleaved apart in a protein when specific
peptide bonds are hydrolysed within the polypeptide chain
 The cleaving of peptide bonds is performed by
proteins called ___________
 Three examples of different types of protease are
 Carboxypeptidase A
 Chymotrypsin
 HIV Protease
 Carboxypeptidase A
 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 ______
 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
 Chymotrypsin
 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 ___________ or large hydrophobic residues
 Figure 9-19, page 253 (9-18, page 273)
 HIV Protease
 HIV Protease is a member of the Aspartic Protease family
 _____________ 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
 Figures 9-20 & 9-21, pages 253 & 254 (9-19, 9-20, p.273, 274)
 The HIV Protease Inhibitors have been produced as
a result
 Preproproteins and Proproteins
 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 _________ the protein
correctly
 The longer chain renders the protein ________
 Example: Chymotrypsin is formed as an inactive
Prochymotrypsin and is only activated when cleaved to
the native chain length
 Summary of Lecture Four:
 PROTEIN MODIFICATION
 ACETYLATION
 Example: Stabilises a protein to degradation
 HYDROXYLATION
 Example: Hydroxyproline
 Stabilises the structure of collagen
 GLYCOSYLATION
 Can make a protein more soluble
 Orientates proteins in membranes
 PHOSPHORYLATION
 Most ubiquitous of modifications
 Integral to enzyme control
 PROTEIN CLEAVAGE
 The hydrolysis of specific peptide bonds
 Performed by enzymes known as Proteases
 Native protein chains can be produced by cleaving longer
chain preproproteins and proproteins
 Needed for the correct transportation, for
inactivation, and for the correct folding of proteins