Aspartate/aspartic acid degradation

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

Joshi AB, Sawai M, Kearney WR, et al. Studies on the mechanism of aspartic acid cleavage and glutamine deamidation in the acidic degradation of glucagon. /. Pharm. Sci. 2005 94 1912-1927. 

L and the D/L ratio approaches zero. After the death of the living organism, proteins start to spontaneously break down. An inter-conversion of the amino acids occurs from one chiral form (L) to a mixture of D- and L- forms following protein degradation this process is called amino acid racemisation. The extent of racemisation is measured by the ratio of D/L isomers and increases as a function of time and temperature. The longer the racemisation process continues the closer to 1 the ratio between the D- and L-forms becomes. If the D/L ratio is <1 it may be possible to use it to estimate age. The D/L ratio of aspartic acid and isoleucine are the most widely used for this dating technique [104]. Dates have been obtained as old as 200 000 years. However, it has been used mainly to date samples in the 5000 100 000 year range. Recent studies [ 105] mention an estimation of the method accuracy to be around 20%. 

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. 

We now examine the degradation of two selected bioactive peptides containing aspartic acid residues flanked by activating residues. 

Asparagine residues (and glutamine residues, see below) are sites of particular instability in peptides. As will be exemplified below, rates of degradation at asparagine residues are markedly faster (tenfold and even much more) than at aspartic acid residues. As reported, the tm values for the internal asparagine in a large number of pentapeptides ranged from 6 to 507 d under... 

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

A contemporaneous study on the same subject utilized a chemical correlation method where (—)-A-benzylargemonine chloride, obtained by sequential optical resolution and quatemization of ( )-7V-methylpavine (5), underwent a multistep degradative process to furnish (-)-A,A-dimethyl-di-H-propyl aspartate. Comparison of this final product with L-aspartic acid of known chirality led to the absolute configuration of (—)-5 (115,158). (—)-Eschscholtzine (9) was assigned the same absolute configuration by correlation of its ORD curve and optical rotation with those of (—)-argemonine (775). 

In general, transition metal ions are undesired in protein formulations because they can catalyze physical and chemical degradation reactions in proteins. However, specific metal ions are included in formulations when they are cofactors to proteins and in suspension formulations of proteins where they form coordination complexes (e.g., zinc suspension of insulin). Recently, the use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (63). 

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