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Chemical Reactivity of Amino Acid Side Chains

Chemical Reactivity of Amino Acid Side Chains

You should be able to identify which side chains contain H bond donors and acceptors. Likewise, some are acids and bases. You should be familiar with the approximate pKa's of the side chains, and the N and C terminal groups. Three of the amino acid side chains (Trp, Tyr, and Phe) contribute significantly to the UV absorption of a protein at 280 nm. This section will be dealing predominantly with the chemical reactivity of the side chains, which is important in understanding the properties of the proteins. Many of the side chains are nucleophiles. Nucleophilicity is a measure of how rapidly molecules with lone pairs of electrons can react in nucleophilic substitution reactions. It correlates with basicity, which measures the extent to which a molecule with lone pairs can react with an acid (Bronsted or Lewis). The properties of the atom which holds the lone pair are important in determining both nucleophilicity and basicity. In both cases, the atom must be willing to share its unbonded electron pair. If the atom holding the nonbonded pair is more electronegative, it will be less likely to share its electrons, and that molecule will be a poorer nucleophile (nu:) and weaker base. Using these ideas, it should be clear that RNH2 is a better nucelophile than ROH, OH- is a better than H2O and RSH is a better than H2O. In the latter case, S is bigger and its electron cloud is more polarizable - hence it is more reactive. The important side chain nucleophiles (in order from most to least nucleophilic) are Cys (RSH, pKa 8.5-9.5), His (pKa 6-7), Lys (pKa 10.5) and Ser (ROH, pKa 13).

An understanding of the chemical reactivity of the various R group side chains of the amino acids in a protein is important since chemical reagents that react specifically with a given amino acid side chain can be used to:

  • identify the presence of the amino acids in unknown proteins or
  • determine if a given amino acid is critical for the structure or function of the protein. For example, if a reagent that covalently interacts with only Lys is found to inhibit the function of the protein, a lysine might be considered to be important in the catalytic activity of the protein.

Figure: A REVIEW SUMMARY OF THE CHEMISTRY OF ALDEHYDES, KETONES, AND CARBOXYLIC ACID DERIVATIVES

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1. Ser: Generally No More Reactive Than Ethanol

It is a potent nucleophile in a certain class of proteins (proteases, for example) when it is deprotonated.

2. Lys: (or N-terminal RNH2) This Is a Potent Nu: Only When Deprotonated

Figure: LYSINE REACTIONS 1

  • reacts with anhydride in a nucleophilic substitution reaction (acylation).
  • reacts reversibly with methylmaleic anhydride (also called citraconic anhydride) in a nucleophilic substitution reaction.
  • reacts with high specificity and yield toward ethylacetimidate in a nucleophilic substitution reaction (ethylacetimidate is like ethylacetate only with a imido group replacing the carbonyl oxygen). Ethanol leaves as the amidino group forms. (has two N -i.e. din - attached to the C)

06lysrx1.gif

Figure: LYSINE REACTIONS 2

  • reacts with O-methylisourea in a nucleophilic substitution reaction with the expulsion of methanol to form a guanidino group (has 3 N attached to C, nidi)
  • reacts with fluorodinitrobenzene (FDNB or Sanger's reagent) or trinitrobenzenesulfonate (TNBS, as we saw with the reaction with phosphatidylethanolamine) in a nucleophilic aromatic substitution reaction to form 2,4-DNP-lysine or TNB-lysine.
  • reacts with Dimethylaminonapthelenesulfonylchloride (Dansyl Chloride) in a nucleophilic substitution reaction.

07lysrx2.gif

Figure: LYSINE REACTIONS 3

  • reacts with high specificity toward aldehydes to form imines (Schiff bases), which can be reduced with sodium borohydride or cyanoborohydride to form a secondary amine.

08lysrx3.gif

3. Cys: A Potent Nucleophile, Which Is Often Linked to Another Cys to Form a Covalent Disulfide Bond

Figure: CYSTEINE REACTIONS 1

  • reacts with iodoacetic acid in an SN2 reaction, adding a carboxymethyl group to the S.
  • reacts with iodoacetamide in an SN2 reaction, adding a carboxyamidomethyl group to S.
  • reacts with N-ethylmaleimide in an addition reaction to the double bond.

09cysrx1.gif

Figure: a quick review of sulfur redox chemistry

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Figure: CYSTEINE REACTIONS 2

  • reacts with R'-S-S-R'', a disulfide, in a disulfide interchange reaction, to form R-S-S-R'.
  • reacts with oxidizing agents like HCOOOH, performic acid, to form cysteic acid.
  • reacts with 5,5-Dithiobis (2-nitrobenzoic acid) (DTNB or Ellman's reagent) in a RSH displacement reaction in which DTNB is cleaved and the 2-nitro-5-thiobenzoic acid anion, which absorbs at 412 nm, is released. Used to quantitate total RSH in a protein.

11cysrx2.gif

Cystine - Disulfides

Two cysteine side chains can covalently interact in a protein to produce a disulfide. Just as HOOH (hydrogen peroxide) is more oxidized than HOH (O in H2O2 has oxidation number of 1- while the O in H2O has an oxidation number of 2-) , RSSR is the oxidized form (S oxidation number 1-) and RSH is the reduced form (S oxidation number 2-) of thiols. Their oxidation numbers are analogous since O and S are both in Group 6 of the periodic table and both are more electronegative than C.

A quick review of redox reactions and oxidation numbers: BC Online; ChemWiki

Figure: DISULFIDE - CYSTINE - REACTIONS

12disulfide.gif

When a protein folds, two Cys side chains might approach each other, and form an intrachain disulfide bond. Likewise, two Cys side chains on separate proteins might approach each other and form an interchain disulfide. Such disulfides must be cleaved, and the chains separated, before analyzing the sequence of the protein. The disulfide in protein can be cleaved by reducing agents such as beta-mercaptoethanol, dithiothreitol, tris (2-carboxyethyl) phosphine (TCEP) or oxidizing agents which further oxidize the disulfide to separate cysteic acids.

Figure: Disulfide Oxidizing Agents - b-mercaptoethanol, dithiothreitol, and phosphines

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Figure: TCEP reduction of disulfides

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The inside of cells are maintained in a reduced environment by the presence of many "reducing" agents, such as the tripeptide γ-glu-cys-gly (glutathione). Hence intracellular proteins usually do not contain disulfides, which are abundant in extracellular proteins (such as those found in blood).

Figure: Cleaving Disulfide Bonds in Proteins

15disulfidebreak.gif

Cysteine Redox Chemistry

The sulfur in cysteine is redox-active and hence can exist in a wide variety of states, depending on the local redox environment and the presence of oxidizing and reducing agents. A potent oxidizing agent that can be made in cells is hydrogen peroxide, which can lead to more drastic and irreversible chemical modifications to the Cys side chains.  If a reactive Cys is important to protein function, then the function of the protein can be modulated (sometimes reversibly, sometimes irreversibly) with various oxidizing agents, as shown in the figure below.

Figure: Redox state of Cysteine

15.1CysChem.gif

4. Histidine: One of the Strongest Bases at Physiological pH's

The nitrogen atom in a secondary amine might be expected to be a stronger nucleophile than a primary amine through electron release to that N in a secondary amine. Opposing this effect is the steric hindrance by the two attached Cs of the N on attack on an electrophile. However, in His, this steric effect is minimized since the 2Cs are restrained by the ring. With a pKa of about 6.5, this amino acid is one of the strongest available bases at physiological pH (7.0). Hence, it can often cross-react with many of the reagents used to modify Lys side chains. His reacts with reasonably high selectivity with diethyl pyrocarbonate.

Figure: REACTIONS OF HISTIDINE

16hisrx.gif

Figure: Where is the H on His? Where is the Charge?

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Amino acids in naturally occurring proteins are also subjected to chemical modification within cells. These modifications alter the properties of the amino acid that is modified, which can alter the structure and function of the protein. Most chemical modifications made to proteins within cells occur after the protein is synthesized in a process called translation. The resulting chemical changes are termed post-translational modifications.

Figure: Post-translational modification of proteins

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Here is a list of post-translational modification from the Swiss Institute of Bioinformatics:

  • PDOC00001 1 N-glycosylation site
  • PDOC00002 1 Glycosaminoglycan attachment site
  • PDOC00003 1 Tyrosine sulfation site
  • PDOC00004 1 cAMP- and cGMP-dependent protein kinase phosphorylation site
  • PDOC00005 1 Protein kinase C phosphorylation site
  • PDOC00006 1 Casein kinase II phosphorylation site
  • PDOC00007 1 Tyrosine kinase phosphorylation site
  • PDOC00008 1 N-myristoylation site
  • PDOC00009 1 Amidation site
  • PDOC00010 1 Aspartic acid and asparagine hydroxylation site
  • PDOC00012 1 Phosphopantetheine attachment site
  • PDOC00013 1 Prokaryotic membrane lipoprotein lipid attachment site
  • PDOC00342 1 Prokaryotic N-terminal methylation site
  • PDOC00266 1 Prenyl group binding site (CAAX box)
  • PDOC00687 2 Intein N- and C-terminal splicing motif profiles