Aspartic acid residues, reactivity

Reactivity of Aspartic Acid Residues 6.3.3.1. Reaction Mechanisms... 

It is a common observation that a peptide bond formed by an aspartic acid residue is cleaved in dilute acids at a rate at least 100-fold faster than other peptide bonds. Three reaction mechanisms are now known to account for this specific reactivity [9] [90—95]. 

Further insights into the influence of pH on the reactivity at aspartic acid residues are provided by a study of the model peptide Val-Tyr-Pro-Asp-Gly-Ala (Fig. 6.28,a) [93], At pH 1 and 37°, the tm value for degradation was ca. 450 h, with cleavage of the Asp-Gly bond predominating approximately fourfold over formation of the succinimidyl hexapeptide. At pH 4 and 37°, the tm value was ca. 260 h due to the rapid formation of the succinimidyl hexapeptide, which was slowly replaced by the iso-aspartyl hexapeptide. Cleavage of the Asp-Gly bond was a minor route. At pH 10 and 37°, the tm value was ca. 1700 h, and the iso-aspartyl hexapeptide was the only breakdown product seen. In Sect. 6.3.3.2, we will compare this peptide with three analogues to evaluate the influence of flanking residues. 

All the evidence presented in this section concerns aspartic acid residues, and one may wonder whether glutamic acid residues display similar reactivity. The answer is clearly that they do not, in particular for entropy reasons. In fact, replacement of a reactive aspartic acid residue by a glutamic acid residue can greatly increase the chemical stability of a peptide. This is exemplified by human epidermal growth factor (hEGF), an important promoter of... 

The major structural factors that influence the reactivity of aspartic acid residues are i) conformational aspects of the peptide, particularly the local flexibility of the peptide chain as dictated by primary, secondary, and tertiary structure, and ii) the amino acid sequence (i.e., the nature of the adjacent residues). Most of the available evidence concerns the influence of adjacent residues, as discussed in this section. 

Before doing so, we briefly examine the influence of conformation and flexibility. Indeed, formation of succinimide is limited in proteins due to conformational constraints, such that the optimal value of the and ip angles (Sect. 6.1.2) around the aspartic acid and asparagine residues should be +120° and -120°, respectively [99], These constraints often interfere with the reactivity of aspartic acid residues in proteins, but they can be alleviated to some extent by local backbone flexibility when it allows the reacting groups to approach each other and, so, favors the intramolecular reactions depicted in Fig. 6.27. When compared to the same sequence in more-flexible random coils, elements of well-formed secondary structure, especially a-helices and 13-turns, markedly reduce the rate of succinimide formation and other intramolecular reactions [90][100],... 

As in the case of degradation at aspartic acid residues, the major structural factors that influence the reactivity of asparagine and glutamine residues... 

The influence of secondary structure on reactions of deamidation has been confirmed in a number of studies. Thus, deamidation was inversely proportional to the extent of a-helicity in model peptides [120], Similarly, a-hel-ices and /3-turns were found to stabilize asparagine residues against deamidation, whereas the effect of /3-sheets was unclear [114], The tertiary structure of proteins is also a major determinant of chemical stability, in particular against deamidation [121], on the basis of several factors such as the stabilization of elements of secondary structure and restrictions to local flexibility, as also discussed for the reactivity of aspartic acid residues (Sect. 6.3.3). Furthermore, deamidation is markedly decreased in regions of low polarity in the interior of proteins because the formation of cyclic imides (Fig. 6.29, Pathway e) is favored by deprotonation of the nucleophilic backbone N-atom, which is markedly reduced in solvents of low polarity [100][112],... 

A more complete but more speculative picture of the reactive site of the lipase (Figure 4) (12) shows the hydrophobic leucine next to the reactive serine. I suggest that the leucine is not buried inside the enzyme but exposed on the surface and held in place by steric restriction or hydro-phobic binding by another amino acid. The leucine could then contribute not only to the hydrophobicity of the reactive site but also to its sterically hindered nature. An aspartic acid residue in chymotrypsin assists in the activation of the nucleophilic serine hydroxyl through a charge relay system. Lipase may have a similar system, and aspartic acid is therefore included in the model (Figure 4). 

Several pharmaceutical enzymes belong to the group of serine-histidine estero-proteolytic enzymes (serine proteases), which display their catalytic activity with the aid of an especially reactive serine residue, whose (3-hydroxyl group forms a covalent bond with the substrate molecule. This reaction takes place by cooperation with the imidazole base of histidine. The specificity of the enzymes is achieved by the characteristic structure of their substrate-binding centers, which in these proteases are built according to the same principle. They consist of a hydrophobic slit formed by apolar side chains of amino acids and a dissociated side chain-located carboxyl group of an aspartic acid residue at the bottom. 

As to the influence of adjacent residues, four amino acids are known to increase the reactivity of aspartic acid. These are glycine, proline, histidine, and serine, as explained and exemplified below [101]. To the best of our knowledge, most of the available evidence concerns the facilitating effects of glycine and proline. 

In the case of the influence of adjacent residues, there are clear mechanistic analogies between activation of aspartic acid (Sect. 6.3.3.2) and asparagine sites. The presence of a C-flanking glycine residue consistently increases deamidation of peptides, for the reasons discussed in Sect. 6.3.3.2 [6], Replacement of glycine with a more bulky residue such as valine, leucine, or proline can decrease reactivity more than tenfold [99]. 

C-Flanking serine or cysteine residues can increase the rate of deamidation and, particularly, cleavage, in analogy with the mechanism discussed for aspartic acid [92], Increased reactivity can also result from the presence of a C-flanking histidine, which increases the nucleophilicity of the Asn side-chain amido group and, thus, favors Pathways f and perhaps d in Fig. 6.29 [124], N-Flanking lysine was also found to facilitate Pathway e (Fig. 6.29) in a pH-dependent maimer, likely by increasing the electrophilicity of the carbonyl C-atom in the Asn side chain [125]. 

The first designed catalyst where there was some understanding of the relationship between structure and function was oxaldie 1, a 14-residue peptide that folds in solution to form helical bundles [11] (Fig. 12). Oxaldie 1 was designed to catalyze the decarboxylation of oxaloacetate, the a-keto acid of aspartic acid, via a mechanism where a primary amine reacts with the ketone carbonyl group to form a carbinolamine that is decarboxylated to form pyruvate. The reaction is piCj dependent and proceeds faster the lower the piC of the primary amine if the reaction is carried out at a pH that is lower than the piCj, of the reactive amine. The sequence contains five lysine residues that in the folded state form... 

In summary, protein molecules may contain up to nine amino acids that are readily derivatizable at their side chains aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, tyrosine, methionine, and tryptophan. These nine residues contain eight principal functional groups with sufficient reactivity for modification reactions primary amines, carboxylates, sulfhydryls (or disulfides), thioethers, imidazolyls, gua-nidinyl groups, and phenolic and indolyl rings. All of these side chain functional groups in addition to the N-terminal a-amino and the C-terminal a-carboxylate form the full complement of polypeptide reactivity within proteins (Fig. 12). 

Proteases are classified according to their catalytic mechanism. There are serine, cysteine, aspartic, and metalloproteases. This classification is determined through reactivity toward inhibitors that act on particular amino acid residues in the active site region of the enzyme. The serine proteases are widely distributed among microbes. The enzymes have a reactive serine residue in the active site and are generally inhibited by DFP or PMSF. They... 

The correlation between racemization rates in free amino acids and the o values also supports the carbanion-intermediate mechanism of racemization (17). The R-group can act to stabilize the negative charge on the a-carbon so that the carbanion intermediate is more stable. Since the a values also agree with the racemization rates observed in the present study, the same mechanism probably operates with protein-bound amino acids. It is noteworthy, however, that the racemization rate of free aspartic acid is 10-5 relative to those reported here for this amino acid residue in proteins (17-19). (For relevant discussions on the influence of R groups on reactivities of amino acids, peptides, and proteins, see references 21-26). 

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