Protein Modification Overview DEFINITION The modification of selected residues in a protein and not as a component of synthesis

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1 Lecture Four: Protein Modification & Cleavage [Based on Chapters 2, 9, 10 & 11 Berg, Tymoczko & Stryer] (Figures in red are for the 7th Edition) (Figures in Blue are for the 8th 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 H 3 C C S CoA + H 2 N Protein O H 3 C 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

2 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 (2-64, page 57) (2-64, page 57) 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 & Figure 11-20, page 316 (11-15 & 11-16, p. 340) (11-15 & 11-16, p. 326) 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

3 Figure 11-21, page 317 (11-17, p 341) (11-17, page 327) 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) (11-15, page 326) 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] [Figures, pages 295,296] 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

4 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 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 Chymotrypsin cleaves peptide bonds on the carboxyl side of or large hydrophobic residues Figure 9-19, page 252 (9-18, page 273) (9-18, page 262) HIV Protease HIV Protease is a member of the Aspartic Protease family residues are central to the active site The first role of HIV Protease is to cleave itself out of a large chain protein formed from the genetic material of the virus

5 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) (9-19, 9-20, p.263) 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