Glutamic acid side-chain reactions

Most proteins contain an abundance of carboxylic acid groups from C-terminal functionalities and aspartic and glutamic acid side chains. These groups are readily modified with bis-hydrazide compounds to yield useful hydrazide-activated derivatives. Both carbohydrazide and adipic acid dihydrazide have been employed in forming these modifications using the carbodi-imide reaction (Wilchek and Bayer, 1987). 

Acid-base catalysis does not contribute to rate enhancement by a factor greater than —100, but together with other mechanisms that operate in the active site of an enzyme, it contributes considerably to increasing the enzymatic rate of reactions. The amino acid side chains of glutamic acid, histidine, aspartic acid, lysine, tyrosine, and cysteine in their protonated forms can act as acid catalysts and in their unprotonated forms as base catalysts (see Prob. 8.11). Clearly, the effectiveness of the side chain as a catalyst will depend on the p/ffl (Chap. 3) in the environment of the active site and on the pH at which the enzyme operates. 

The other commonly occurring amino acid with an acidic side-chain is glutamic acid. This compound is probably best known as its monosodium salt (monosodium glutamate or MSG). This salt is added to foods (especially oriental food) to enhance the flavour and impart a meat-like taste to the food. Interestingly, both the d enantiomer of glutamic acid and the naturally occurring l form are used as food additives. Use of the nonnatural d isomer may account for some of the adverse reactions experienced by consumers of MSG in food. 

The side chains of the 20 different amino acids listed in Panel 1.1 (pp. 6-7) have very different chemical properties and are utilized for a wide variety of biological functions. However, their chemical versatility is not unlimited, and for some functions metal atoms are more suitable and more efficient. Electron-transfer reactions are an important example. Fortunately the side chains of histidine, cysteine, aspartic acid, and glutamic acid are excellent metal ligands, and a fairly large number of proteins have recruited metal atoms as intrinsic parts of their structures among the frequently used metals are iron, zinc, magnesium, and calcium. Several metallo proteins are discussed in detail in later chapters and it suffices here to mention briefly a few examples of iron and zinc proteins. 

In contrast to the lability of certain dN adducts formed by the BHT metabolite above, amino acid and protein adducts formed by this metabolite were relatively stable.28,29 The thiol of cysteine reacted most rapidly in accord with its nucleophilic strength and was followed in reactivity by the a-amine common to all amino acids. This type of amine even reacted preferentially over the e-amine of lysine.28 In proteins, however, the e-amine of lysine and thiol of cysteine dominate reaction since the vast majority of a-amino groups are involved in peptide bonds. Other nucleophilic side chains such as the carboxylate of aspartate and glutamate and the imidazole of histidine may react as well, but their adducts are likely to be too labile to detect as suggested by the relative stability of QMs and the leaving group ability of the carboxylate and imidazole groups (see Section 9.2.3). 

The most significant amino acids for modification and conjugation purposes are the ones containing ionizable side chains aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, and tyrosine (Figure 1.6). In their unprotonated state, each of these side chains can be potent nucleophiles to engage in addition reactions (see the discussion on nucleophilicity below). 

---