3/19/10 1 Post translational modification in proteins Manickam Sugumaran Professor of Biology U.Mass/Boston Boston, MA 02125 Post Translational Modification Amino acids Aminoacyl tRNA Modified Amino acids Modified Aminoacyl tRNA Nascent Peptide Nascent Peptide Undergoing Modification Matured Protein Modified Matured Protein
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Post translational modification in proteinsfaculty.umb.edu/yvonne_vaillancourt/Biology/Protein Chem...3/19/10 2 First modification - Modification of N-terminal met. AUG is the universal
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First modification - Modification of N-terminal met. ♦ AUG is the universal Starting codon. ♦ Hence, Met is the first amino acid in all proteins. ♦ But met has to be cleaved. ♦ It occurs as and when the protein is being
synthesized. ♦ Eukaryotes: Removal of Met to expose new amino terminal. ♦ Prokaryotes:
A) Removal of Formyl group. B) Removal of formyl met group.
First Post translational modification ♦ N-terminal Met (or formylmet) of all proteins are
bone calcification. ♦ Methylation - bacterial chemotaxis. ♦ ADP-ribosylation in histones. ♦ γ-glutamyl cysteine formation - α2-macroglobulin. ♦ γ-glutamyl serine - in proline reductase. ♦ Silent modification - Conversion to gln.
In some yeast, Glu-tRNA is modified to Gln-tRNA (There is no charging enzyme for Gln).
γ-Carboxyglutamic acid formation
COOH
Glutamic acid
COOH
COOH
Vitamin K
γ-Carboxyglutamic acid
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Asparagine and Glutamine
H2N COOH
CONH2
H2N COOH
CONH2
Asparagine Glutamine
Modifications of Asparagine 1. Asparagine linked oligosaccharide
formation is a major modification of this amino acid.
2. Isopeptide bond formation with ε-amino group of lysine in peptides.
3. Silent modification to aspartic acid. Neutral amino acid to acidic amino acid formation in proteins.
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Asparagine - Oligosaccharide addition.
Isopeptide bond formation -Both Asn and Gln undergo this reaction.
CONH2
Asparagine
NH2
Lysine Isopeptide
NH
COTransglutaminase
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Silent modification -Both Asn and Gln are converted to carboxylic acids by this mechanism.
HN CO
CONH2
AsparagineHN CO
COOH
Aspartic acid
Modifications of Serine and Threonine
Both these amino acids have the same β-hydroxy group. Therefore, they show the same type of
modifications
COOHH2N
OH
COOHH2N
OH
Serine Threonine
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Serine and Threonine
A. O- linked oligosaccharide formation. B. Phosphorylation -
¨ Regulation of enzyme activity. C. Phosphodiester formation ¨ ser-P-ser and ser-P-pantetheinine. ¨ ADP- Ribosylation. ¨ carbohydrate-P- ser D. Other possible modifications
phosphate addition in proteins. ♦ Acylation - phosphate, acetyl, N-methylalanyl,
dimethyl pimelyl, biotinyl, lipoyl, glutamyl and aspartyl groups (the last two in isopeptide bond).
♦ Methylation - mono, di and tri methyl lysines.
Lysine undergoes hydroxylation in protein to form δ-hydroxylysine
NH2NH2
HOHydroxylation
Collagen Lysine Hydroxylation
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Lysyl oxidase converts lysine to allysine in proteins. This initiates a
number of crosslink formation
Lysyl oxidase
NH2O
Lysine Allysine
Lysine Crosslinks in Elastin
NH2 O
Lysine Allysine
NH
Lysinonorleucine
Fourlysines
Desmosine
N
RR
R
R
+
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Collagen Crosslinks
NH2 H2N
O O
Lysine
Lysyl Oxidase
Allysine
CHOAldol formation
Aldol crosslink
N
R
R OH
R
OH
+
Hydroxypyridiniumcrosslink(Pyridinoline)
Two Hydroxylysineand one lysine
Schiff’s Base formation Both retinal and pyridoxal phosphate are bound to the protein lysines by schiff’s base formation.
NH2
Lysine
N
OH
OH
CH3
OP
O
OO
+H
Pyridoxal phosphate PALP-Lysine adduct
N
OH
H
CH3
OP
O
OO
+H
N
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OAll - trans - RETINAL
HN
NHO
OH2N
LYSINE
HN
NHO
ONH
hνHN
NHO
ON
All - trans - RETINAL PROTONATED SCHIFF'S BASE
13 - cis - RETINAL DEPROTONATED SCHIFF'S BASE
Coenzyme addition by acylation
S SNH
O
SNH
O
HN NH
O
Lipoyl - Lysine Adduct
Biotinyl - Lysine Adduct
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Hydroxylysine undergoes the same modifications as lysine
In addition, it also undergoes carbohydrate addition on the newly formed hydroxyl group.
Proline Modifications 4-Hydroxyproline and 3-hydroxyproline (to some extent 3,4-dehydroproline) formation by protein proline hydroxylases is the major modification. The resultant hydroxyl group is the site of attachment of carbohydrates in some proteins.
Hydroxylation
Collagen Proline Hydroxylation
N N
OH
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Cysteine modifications Important modification is disulfide formation to Cystine
Tyrosine Hydroxylation Although tyrosinase performs this reaction, the existence of a separate hydroxylase cannot be
ruled out in some cases.
OHPeptidyl tyrosine Peptidyl dopa
OH
OH
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Dityrosine formation Peroxidase is responsible for this reaction
Peptidyl tyrosines
+
Isodityrosine cross link dityrosine cross link
OH
OHOH
OHOH
O
Topaquinone as a new cofactor in plasma amine oxidase
OHPeptidyl tyrosine Peptidyl dopa
OH
OH
O
O
OH
OH
HO
Peptidyl dopaquinone
Peptidyl topaPeptidyl topaquinone
OH
O
O
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Modification of Tryptophan An enzyme hydroxylating trp also attacks
peptidyl trp. So the possibility exists.
NH H
N
OH
Peptidyl tryptophan Hydroxytryptophan
HN N
O
H
Dehydro tryptophan Keto tryptophan
Amino acids that do not have any post translational modification
H2N COOH
H
H2N COOH
CH3
H2N COOH
H2N COOH
GLY ALA VAL
LEU ILE
H2N COOH
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Nonspecific modification of all modifiable amino acids
(Bulk of the work was from our lab) In insects as well as most other arthropods, hardening of their exoskeleton is achieved by nonspecific arylation of all modifiable amino acids. The mechanisms are shown in next slide. It is vital for the survival of most arthropods.
CATECHOLS
REACTIVEINTERMEDIATES
PROTEINSCHITIN
SCLEROTIZED CUTICLE
PHENOLOXIDASE
HO
HO
N CH3
O
H
O
O
N CH3
O
HHO
O
N CH3
O
HO
HO
N CH3
O
H
HO
HO
N CH3
O
H
HO
O
N CH3
O
H
O
O
N CH3
O
HHO
HO
N CH3
O
H
HO
HO
N CH3
O
H
N-ACETYLDOPAMINE (NADA)
NADA QUINONE
NADA QUINONE METHIDE
QUINONE METHIDE IMINE AMIDE
DEHYDRO NADA QUINONE
DEHYDRO NADA
QUINONE TANNING
A
BC
A
D
D
D
D
UNIFIED MECHANISM FOR SCLEROTIZATION
QUINONE METHIDE TANNING
QUINONE METHIDE TANNING
A = PHENOLOXIDASE; B = QUINONE ISOMERASE C = QUINONE METHIDE ISOMERASE; D = NONENZYMATIC REACTIONS