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Home , Carboxyglutamic acid, Desmosine, Formylation, Hydroxylysine, Pyroglutamic acid

3/19/10

Post translational modification in proteins

Manickam Sugumaran Professor of Biology U.Mass/Boston Boston, MA 02125

Post Translational Modification

Amino acids Modified Amino acids

Aminoacyl tRNA Modified Aminoacyl tRNA

Nascent Peptide Nascent Peptide Undergoing Modification

Matured Protein Modified Matured Protein

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First modification - Modification of N-terminal met.

♦ AUG is the universal Starting codon. ♦ Hence, Met is the first 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

trimmed first to generate a new N-terminus.

Proteolytic cleavage

COOH

H2N Met

COOH

H2N

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PROCESSING OF INSULIN

LEADER 2 1 SEQUENCE B CHAIN 23 aa

M CONNECTING NH2 SEQUENCE

COOH COOH COOH A CHAIN PREPROINSULIN FORMATION

A CHAIN 30 aa 21 aa 33 aa COOH COOH CONNECTING B CHAIN SEQUENCE INSULIN PROINSULIN

N-Terminal Acylation.

♦ . ♦ . ♦ Pyruvoyl formation. ♦ α-ketobutyryl formation. ♦ Glucuronylation. ♦ α-amino acylation. ♦ Pyroglutamyl formation. ♦ Murein addition.

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Pyruvoyl group formation at N- terminal of some enzymes.

N-Terminal is specifically converted to in some proteins.

HOOC Enzyme O H N N 2 H N-Terminal Glutamic acid Pyroglutamic acid

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N-Terminal N-terminal glutamine undergoes spontaneous cyclization with the elimination of ammonia to form pyroglutamic acid.

H2NCO O H N N 2 H N-Terminal Glutamine Pyroglutamic acid

N-terminal alkylation

♦ : High concentration of glucose in blood results in glycosylation of aminoterminal valine in hemoglobin to give 1-deoxy,1-(N-α-valine)- fructose. ♦ N- of some ribosomal proteins at met, ala, and pro.

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Glycosylation of Hemoglobin

NH2

N O H H NH

OH OH O HO HO HO OH 1 OH 2 OH OH OH OH

CH2OH CH2OH CH2OH Glucose Hemoglobin Hemoglubin Schiff's Base -Fructose

♦ 1. Schiff’s base formation. ♦ 2. Amadori rearrangement.

C-Terminal Processing

In some proteins, C-terminal peptide is cleaved Much like the N-terminal signal processing.

 Three modifications of C-terminal. ¨ 1. Conversion of COOH to CONH2 ¨ 2. ADP-Ribosylation of C-terminus in H1. ¨ 3. Substituted formation by addition of to C-terminal carboxyl group.

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Transformations of

1. Methylation. H N NH (Mono & dimethyl) 2 NH 2. ADP-ribosylation 3. 4. formation 5. formation Arginine

Ornithine formation in some proteins is probably

catalyzed by Arginase

H2N NH Arginase NH NH2

NH2 H2O H2N O Arginine Urea Ornithine

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Aspartic acid and glutamic acid

COOH COOH

H2N COOH H2N COOH Glutamic acid

Aspartic acid

1. Phosphorylation - in ATPases 2. Methylation 3. Slow - Approximately 0.1% of Asp in some proteins undergo spontaneous racemization. - Useful for dating proteins found in fossils.

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Modifications of Glutamic acid

♦ γ-carboxyglutamic acid formation - blood clotting, bone calcification. ♦ Methylation - bacterial chemotaxis. ♦ ADP-ribosylation in . ♦ γ-glutamyl formation - α2-macroglobulin. ♦ γ-glutamyl - in 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

COOH COOH

Glutamic acid γ-Carboxyglutamic acid

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Asparagine and Glutamine

CONH2 CONH2

H2N COOH H2N COOH Glutamine

Modifications of Asparagine

1. Asparagine linked oligosaccharide formation is a major modification of this amino acid. 2. 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.

Asparagine

CO CONH 2 NH2 NH

Lysine Isopeptide

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Silent modification -Both Asn and Gln are converted to carboxylic acids by this mechanism.

CONH2 COOH

HN CO HN CO Asparagine Aspartic acid

Modifications of Serine and Both these amino acids have the same β-hydroxy group. Therefore, they show the same type of modifications

OH OH

H2N COOH H2N COOH 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 ¨ (methylation and esterification)

Modifications of 1. Phosphorylation (N) 2. Methylation (N) 3. Iodination (4C) 4. Flavin addition (N) 5. ADP-ribosylation (N)

HN N

Histidine

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Lysine modifications

♦ Lysyl - production. ♦ Lysyl oxidation (conversion to ). ♦ Glycosylation (hemoglobin) (Seen already). ♦ Schiff’s base formation - retinal, pyridoxal 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 .

Lysine undergoes hydroxylation in protein to form δ-hydroxylysine

NH NH2 2 HO Hydroxylation

Collagen Lysine Hydroxylation

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Lysyl oxidase converts lysine to allysine in proteins. This initiates a number of crosslink formation

O NH2

Lysine Allysine

Lysine Crosslinks in

R

NH O 2 R R Lysine Allysine + N Four lysines NH

Lysinonorleucine R

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Collagen Crosslinks Lysine

NH2 H2N Two Hydroxylysine and one lysine Lysyl Oxidase R O O R OH + Allysine N Aldol formation Hydroxy OH CHO crosslink (Pyridinoline) R Aldol crosslink

Schiff’s Base formation Both retinal and pyridoxal phosphate are bound to the protein lysines by schiff’s base formation.

Lysine NH 2 H N H O O O O P OH O P OH O O O O + + N CH3 N CH3 H H Pyridoxal phosphate PALP-Lysine adduct

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HN O

OH2N NH All - trans - RETINAL LYSINE O

HN O H N NH O All - trans - RETINAL PROTONATED SCHIFF'S BASE

hν HN O N NH O 13 - cis - RETINAL DEPROTONATED SCHIFF'S BASE

Coenzyme addition by acylation

O S N H HN NH Biotinyl - Lysine Adduct O

O

N S S H Lipoyl - 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- 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.

OH Hydroxylation N N

Collagen Proline Hydroxylation

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Cysteine modifications Important modification is

disulfide formation to Cystine

Oxidation SH S + SH S

Cysteine Cystine

Cysteine - Other modifications

♦Glycosylation ♦Heme addition ♦Flavin addition ♦Phycocyanobilin addition ♦Thiohemiacetal formation ♦Dehydroalanine formation

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Dehydroalanine formation

O(S)H

Serine/Cysteine Dehydroalanine

Methionine modification - Possible reaction

S S O OH

+ O OH

Benzoquinone

Peptidyl Methionine adduct

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Modification of Phenylalanine

HO

Peptidyl -β-hydroxy phenylalanine

Peptidyl phenylalanine ? OH Peptidyl tyrosine

Modification of Tyrosine

♦ Hydroxylation (ring) (and possibly side chain). ♦ Dityrosine formation (ring, O). ♦ Halogenation (ring). ♦ (O). ♦ Phosphorylation (O). ♦ Adenylation and Uridinylation. ♦ Flavin addition (O).

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Halogenation of Tyrosine Simple Halogenation: 1. 3-chloro (bromo, or iodo) 2. 3,5-dichloro (bromo, or iodo) tyrosines. 3. 3-chloro, 5-bromo tyrosine.

♦ Complex halogenation: (hormone) ♦ 1.3,5,3’-triiodothyronine ♦ 2.3,5,3’,5’-tetraiodothyronine (thyroxin)

Tyrosine Hydroxylation Although tyrosinase performs this reaction, the existence of a separate hydroxylase cannot be ruled out in some cases.

OH

OH OH Peptidyl tyrosine Peptidyl dopa

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Dityrosine formation Peroxidase is responsible for this reaction

OH O OH OH

OH + OH

Peptidyl tyrosines Isodityrosine cross link dityrosine cross link

Topaquinone as a new cofactor in plasma amine oxidase

OH O

OH OH O Peptidyl tyrosine Peptidyl dopa Peptidyl dopaquinone

O OH

O OH HO OH

Peptidyl topaquinone Peptidyl topa

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Modification of An enzyme hydroxylating trp also attacks

peptidyl trp. So the possibility exists.

OH

N N H H Peptidyl tryptophan Hydroxytryptophan

O

N N H H Dehydro tryptophan Keto tryptophan

Amino acids that do not have any post translational modification

H CH3

H2N COOH H2N COOH H2N COOH GLY ALA VAL

H2N COOH H2N COOH LEU ILE

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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 CATECHOLS other arthropods, hardening PHENOL of their exoskeleton is OXIDASE achieved by REACTIVE nonspecific arylation of all INTERMEDIATES modifiable amino acids. The mechanisms are shown in CHITIN PROTEINS next slide. It is vital for the SCLEROTIZED survival of most arthropods. CUTICLE

H H H HO N CH3 A O N CH3 D HO N CH3 O O O HO O HO

N-ACETYLDOPAMINE NADA QUINONE QUINONE TANNING (NADA) B H H H HO N CH HO N CH HO N CH3 C 3 D 3 O O O HO O HO

DEHYDRO NADA NADA QUINONE METHIDE QUINONE METHIDE TANNING A H H HO N CH O N CH3 D HO N CH3 D 3 O O O O O HO DEHYDRO NADA QUINONE QUINONE METHIDE QUINONE METHIDE IMINE AMIDE TANNING UNIFIED MECHANISM FOR SCLEROTIZATION A = PHENOLOXIDASE; B = QUINONE ISOMERASE C = QUINONE METHIDE ISOMERASE; D = NONENZYMATIC REACTIONS

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