Modification of amino acid side chains

Transition metals such as iron can catalyze oxidation reactions in aqueous solution, which are known to cause modification of amino acid side chains and damage to polypeptide backbones (see Chapter 1, Section 1.1 Halliwell and Gutteridge, 1984 Kim et al., 1985 Tabor and Richardson, 1987). These reactions can oxidize thiols, create aldehydes and other carbonyls on certain amino acids, and even cleave peptide bonds. The purposeful use of metal-catalyzed oxidation in the study of protein interactions has been done to map interaction surfaces or identify which regions of biomolecules are in contact during specific affinity binding events. 

As described for lEC, elution is done by a stepwise or a continuous change of buffer composition. The mildest elution buffer is an aqueous buffer with low ionic strength, e.g., 20 mm Tris-HCl. If it is not successful, desorb with a chaotropic solvent, e.g., 2 M potassium rhodanide (thiocyanate), 2.5 M guanidinium hydrochloride, up to 7 M urea, or with increasing concentrations of methanol or acetonitrile. Especially the use of rhodanide or urea may be accompanied by a chemical modification of amino acid side chains, which disturbs amino acid analysis. 

Many of radioactive isotopes are very useful for the following biochemical processes (Table 6.1). The radioactive label is introduced into macromolecules, especially proteins, either during biosynthesis, e.g., during translation in the presence of S-methionine, or enzymatically, e.g., by use of P-labeled ATP during protein phosphorylation by protein kinases, or chemically by modification of amino acid side chains. Examples for reagents used in chemical radiolabeling of proteins are given in Table 6.2. 

The presence of O2 causes an increase in the destruction of amino acids and in the amount of amide nitrogen formed and also influences the modification of amino acid side chains. Thus, a major product from PGA irradiated in 02 is aspartic acid, which is formed only in traces in the absence of O2. The inversion of glutamate residues is reduced in O2. 

Lundblad, R. (1996) Chemical modification of amino acid side-chains, in Proteins Labfax (Pace,... 

Figure 3.59. Finishing Touches. Some common and important covalent modifications of amino acid side chains are shown.

Although aggregation is the predominant means by which proteins become inactivated during refolding, several other inactivation pathways have also been observed. Proteins can be inactivated by thiol-disulfide exchange or alteration of the primary structure by chemical modification of amino acid side chains. In addition, refolded proteins may be inactivate due to the absence of prosthetic groups and metals or because of improper association of the subunits in multimeric proteins (79). 

All the changes to proteins that have been described so far are based on the modification of amino acid side-chains in proteins, but of course the protein structure is also influenced by oxidation processes. 

We have seen how allosteric control can provide feedback regulation to maintain order in the body and respond to its needs. Allosteric control is reversible and the analogous type of regulation by covalent modification is the reversible modification of amino-acid side chains. Some enzymes are affected directly by both types of control. By far the most common modification of proteins is the phosphorylation of serine and threonine residues, and tyrosine phosphorylation is a key part of many control mechanisms. Protein phosphorylation is discussed below. Many other types of protein modification occursome reversible and some irreversible. 

Attempts have been made to study the active site by chemical modification of amino acid side chains (Messner e/ aL, 1970 Thrasher / a/., 1975 Thrasher and Cohen, 1971). No attempts have been made to separate the various products of the modification reactions and to study the individual homogeneous populations of modified proteins. Collectively, however, the results of these studies would appear to implicate an amino group in the cytophilic site. The data of Ciccimarra et al. (1975) suggest that there are two lysine residues in a decapeptide containing the site, but the positions of the modified amino groups have not been ascertained, nor has the effect of these reagents on other side chains and on conformations been studied. 

PR Gratzer, J.P Santerre, and J.M. Lee, Modulation of collagen proteolysis by chemical modification of amino acid side-chains in acellularized arteries. Biomaterials, 25 (11), 2081-2094, 2004. 

Apart from the modification of amino acid side chains in individual protein strands, cross-linkage of two protein chains can also occur. Some of the structures are shown in Formula 4.100. Pentosidine was first found in physiological protein. It strongly fluoresces and is formed by bridging an arginine residue with a lysine residue via a pentose. The concentrations of pentosidine in food are comparatively low (Table 4.16). The formation of pentosidine is assumed to be as shown in Formula 4.101. Formation of the Amadori product with the 8-amino group of lysine is followed by water elimination at C-2 and C-3 of pentose with the formation of the 4,5-diulose, which condenses with the... 

Chemical modification of amino acid side chain functionalities will also serve to cleave specific peptide bonds selectively. Chemical cleavage of a polypeptide chain exploits the unique reactivity of chemically modified side chains of particular amino acids in the labilization of adjacent peptide bonds by neighbouring group participation (68). The residues investigated so far for this purpose have been methionine, cysteine and the aromatic amino acids including tryptophan (438-440, 443). 

Chemical modification of amino acid side chains in enzymes has been carried out to induce changes in the biological activity, in the conformation and the physical properties of an enzyme or to introduce special-purpose groups into a protein ( 7, 82, 146, 169, 183, 230, 375, 413). 

---