Identification of isoaspartyl-containing sequences in peptides and proteins that are usually poor substrates for the class II protein carboxyl methyltransferase

We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo.     

New proteomic developments to analyze protein isomerization and their biological significance in plants

Spontaneous isoaspartyl formation from aspartyl dehydration or asparaginyl deamidation is a major source of modifications in protein structures. In cells, these conformational changes could be reverted by the protein L-isoaspartyl methyltransferase (PIMT) repair enzyme that converts the isoaspartyl residues into aspartyl. The physiological importance of this metabolism has been recently illustrated in plants. Recent developments allowing peptide isomer identification and quantification at the proteome scale are portrayed. The relevance of these new proteomic approaches based on 2-D electrophoresis or electron capture dissociation analysis methods was initially documented in mammals. Extended use to Arabidopsis model systems is promising for the discovery of controlling mechanisms induced by these particular post-translational modifications and their biological role in plants.     

Enzymatic methylation of L-isoaspartyl residues derived from aspartyl residues in affinity-purified calmodulin. The role of conformational flexibility in spontaneous isoaspartyl formation

We have investigated the formation of D-aspartyl and L-isoaspartyl (beta-aspartyl) residues and their subsequent methylation in bovine brain calmodulin by the type II protein carboxyl methyltransferase. Based on the results of studies with unstructured peptides and denatured proteins, it has been proposed that the major sites of carboxyl methylation in calmodulin are at L-isoaspartyl residues that originate from two Asn-Gly sequences. To test this hypothesis, we directly identified the sites of methylation in affinity-purified preparations of calmodulin by peptide mapping using the proteases trypsin, endoproteinase Lys-C, clostripain, chymotrypsin, and Staphylococcus aureus V8 protease. We found, however, that the major high-affinity sites of methylation originate from aspartyl residues at position 2 and at positions 78 and/or 80. The methylatable residue in the first case was shown to be L-isoaspartate by comparison of the properties of a synthetic peptide corresponding to the N-terminal 13 residues substituted with an L-iso-Asp residue at position 2. The second methylatable residue, probably derived from Asp78, also appears to be an L-isoaspartyl residue. These sites appear to be readily accessible to the methyltransferase and are present in relatively flexible regions of calmodulin that may allow the spontaneous degradation reactions to occur that generate L-isoaspartyl residues via succinimide intermediates. Interestingly, the four calcium binding regions, each containing 3-4 aspartyl and asparaginyl residues (including the two Asn-Gly sequences), do not appear to contribute to the high-affinity methyl acceptor sites, even when calcium is removed prior to the methylation reaction. We propose that methylatable residues do not form at these sites because of the inflexibility of these regions when calcium is bound.     

Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides

The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects.     

Enzymatic methyl esterification of a deamidated form of mouse epidermal growth factor

The enzyme S-adenosylmethionine:protein carboxyl-O-methyl-transferase, type II (EC 2.1.1.77; PCMT) from eukaryotes methyl esterifies peptides containing isoAsp residues, which can arise from spontaneous deamidation of labile Asn residues. We report here a study on in vitro methyl esterification of mouse EGF by bovine brain PCMT. This peptide contains two Asn in the sequences Asn1-Ser2 and Asn16-Gly17. It is known from the literature that the presence of a small residue on the carboxyl side of asparaginyl makes this residue susceptible to deamidation through the spontaneous formation of a succinimide intermediate. Therefore EGF was incubated under deamidating conditions (pH 9.0, 37 degrees for 48 h) and the extent of deamidation monitored by enzymatically measuring the NH3 produced during the alkali treatment: a release of 0.80 mol NH3/mol EGF was calculated. The alkali-treated EGF, analyzed by anion-exchange chromatography, shows two major components identified as native EGF (nEGF) and its deamidated form (dEGF). When incubated in the presence of purified PCMT neither nEGF nor dEGF showed any methyl accepting capability. Since it is known that the three-dimensional structure of a protein may hinder the methyl esterification of a potential ethyl accepting site, dEGF was unfolded by reducing and alkylating the intrachain disulfide bridges. Only a slight increase in the methyl accepting capability could be observed. Conversely, when EGF was deamidated after its unfolding, the resulting protein was stoichiometrically methylated by PCMT, presumably at level of isoAsp16. Our findings strongly suggest that the three-dimensional structure of a protein is a major specificity determinant for both deamidation and methyl esterification processes.     

Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae

The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.     

Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone

In vitro aging at pH 7.4, 37 degrees C causes natural sequence recombinant human growth hormone (rhGH), methionyl rhGH, and human pituitary growth hormone to become substrates for bovine brain protein carboxyl methyltransferase, an enzyme that modifies the "side chain" alpha-carboxyl group present at atypical isoaspartyl linkages. The substrate capacity of rhGH increased at a rate of 1.8 methyl-accepting sites/day/100 molecules of hormone. Reversed-phase high performance liquid chromatography (HPLC) of trypsin digests of aged rhGH revealed two altered peptides not present in digests of control rhGH. These two fragments, which had the amino acid compositions of residues 128-134 (Leu-Glu-Asp-Gly-Ser-Pro-Arg) and 146-158 (Phe-Asp-Thr-Asn-Ser-His-Asn-Asp-Asp-Ala-Leu-Leu-Lys), contained the majority of the induced methylation sites, 22 and 58%, respectively. Isoaspartate can result from deamidation of asparagine or isomerization of aspartate. Isomerization of Asp-130, the only candidate site in 128-134, was corroborated by coelution of the altered fragment with the synthetic isoaspartyl peptide upon reversed-phase HPLC. Evidence is presented that the altered 146-158 fragment is a mixture of two peptides resulting from deamidation of Asn-149 to form 70-80% isoaspartate and 20-30% aspartate at this position. The position of isoaspartate in the altered 146-158 fragment was deduced from mass spectrometry, which indicated a single deamidated asparagine; from methylation stoichiometry, which indicated only one methylation site; and from automated Edman degradation, which showed an absence of asparagine and a low yield of aspartate at position 149. These results show that isoaspartate formation from both aspartate and asparagine is a significant, and possibly the major, source of spontaneous covalent alteration of rhGH and that enzymatic carboxyl methylation provides a powerful tool for assessing this type of modification.     

Modification of synthetic peptides related to lactate dehydrogenase (231-242) by protein carboxyl methyltransferase and tyrosine protein kinase: effects of introducing an isopeptide bond between aspartic acid-235 and serine-236

The possibility that isoaspartyl residues contribute to the substrate specificity of eucaryotic protein carboxyl methyltransferases and/or tyrosine protein kinases has been investigated with two synthetic oligopeptides, Lys-Gln-Val-Val-Asp/isoAsp-Ser-Ala-Tyr-Glu-Val-Ile-Lys, which correspond to amino acids 231-242 of lactate dehydrogenase. One version of the peptide contains the normal amino acid sequence of the chicken muscle M4 isozyme. The other version contains an isoaspartyl residue in position 235 in place of the normal aspartyl residue; i.e., Asp-235 is linked to Ser-236 via its side-chain beta-carboxyl group, rather than via the usual alpha-carboxyl linkage. The normal peptide corresponds to the sequence around Tyr-238 that is phosphorylated in Rous sarcoma virus infected chick embryo fibroblasts [Cooper, J. A., Esch, F. S., Taylor, S. S., & Hunter, T. (1984) J. Biol Chem. 259, 7835]. Using protein carboxyl methyltransferase purified from bovine brain, we found that the normal peptide did not serve as a methyl-accepting substrate but that the isopeptide served as an excellent substrate, exhibiting a stoichiometry of one methyl group per peptide and Km of 0.54 microM. With tyrosine protein kinase partially purified from normal rat spleen both peptides were found to serve as phosphate acceptors at Tyr-238, exhibiting Km values of 4.7 and 8.9 mM for the normal and isopeptide versions, respectively. These results support the idea that protein carboxyl methyltransferase selectively methylates the alpha-carboxyl group of atypical isoaspartyl residues. In contrast, the presence of isoaspartate had a modest negative effect on substrate activity for a tyrosine protein kinase from rat spleen.     

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions have been studied in synthetic peptides to model these spontaneous processes that alter protein structure and function. We show here that the peptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala undergoes a rapid deamidation reaction with a half-life of only 1.4 days at 37 degrees C, pH 7.4, to give an aspartyl succinimide product. Under these conditions, the succinimide product can further react by hydrolysis (half-time, 2.3h) and by racemization (half-time, 19.5 h). The net product of the deamidation reaction is a mixture of L- and D-normal aspartyl and beta-transpeptidation (isoaspartyl) hexapeptides. Replacement of the asparagine residue by an aspartic acid residue results in a 34-fold decrease in the rate of succinimide formation. Significant racemization was found to accompany the deamidation and isomerization reactions, and most of this could be accounted for by the rapid racemization of the succinimide intermediate. Replacement of the glycyl residue in the asparagine-containing peptide with a bulky leucyl or prolyl residue results in a 33-50-fold decrease in the rate of degradation. Peptide cleavage products are observed when these Asn-Leu and Asn-Pro-containing peptides are incubated. Our studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively.     

Identification of a site for carboxyl methylation in human alpha-globin

The human erythrocyte protein carboxyl methyltransferase modifies unusual protein D-aspartyl and L-isoaspartyl residues which arise spontaneously from internal rearrangements accompanying asparaginyl deamidation and aspartyl isomerization. A site of methylation associated with alpha-globin in intact cells has been identified by peptide mapping of radiolabeled globin isolated from human erythrocytes previously incubated with L-[methyl-3H]methionine. The site is located in a Staphylococcus V8 peptide containing residues 1-30 of alpha-globin. Two potential sources of methylation sites are present in this sequence at Asp-t and Asn-9.     

Role of peptide conformation in the rate and mechanism of deamidation of asparaginyl residues

The tetrapeptides Val-Asn-Gly-Ala and N-acetyl-Val-Asn-Gly-Ala undergo deamidation of the asparaginyl residue at pH 7.0 at similar rates. However, they form different products. The N-acetyl peptide gave a 3:1 ratio of N-acetyl-Val-isoAsp-Gly-Ala and N-acetyl-Val-Asp-Gly-Ala, respectively. The nonacetylated peptide gave no detectable amounts of these products but rather gave a cyclic peptide formed from the nucleophilic displacement of the asparaginyl side chain amide by the amino terminus of valine. This compound was slowly inverted at carbon 2 of the asparaginyl residue. At pH values above 7.5, the nonacetylated peptide also underwent deamidation to form Val-isoAsp-Gly-Ala and Val-Asp-Gly-Ala in the 3:1 ratio. Proton NMR spectra of the acetylated and nonacetylated tetrapeptides show that below pH 7.5 they have very different preferred conformations, and it is these different conformations which result in the different mechanisms of deamidation. Above pH 9.0, both peptides have similar conformations and deamidate by the same mechanism to give equivalent products. Neither mechanism of deamidation was subject to general base catalysis by the buffer. These results suggest that deamidation rates of the asparaginyl-glycyl sequence in proteins will vary according to the conformation of the peptide backbone of each respective protein. The results also show that asparaginyl residues which are penultimate to the amino terminus can react to form an N-terminal-blocked seven-membered ring.     

Selective deamidation and enzymatic methylation of seminal ribonuclease

Isoenzymatic forms alpha 2, alpha beta, and beta 2 of bovine seminal ribonuclease are generated by the transformation of beta-type into alpha-type subunit through deamidation of a single amide group [Di Donato, A., & D'Alessio, G. (1981) Biochemistry 20, 7232-7237]. The residue involved in this selective deamidation has been identified as Asn67. Deamidation occurs by formation of a cyclic imide intermediate involving the Gly at position 68. Opening of the cyclic imide may occur on either side of the nitrogen, generating both the normal alpha-aspartyl and an isoaspartyl residue at position 67. The alpha-carboxyl of the isoaspartyl residue is effectively methylated by bovine brain protein carboxylmethyltransferase.     

Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate. Detection of the reaction products by fast-atom-bombardment mass spectrometry

Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595-602], while very little is known concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11,503-11,511]; (2) the isoaspartyl residue is not recognized unless its alpha-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methyl-esterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.     

Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.     

Replacement of a labile aspartyl residue increases the stability of human epidermal growth factor

Long-term storage of recombinant human epidermal growth factor (EGF), an important promoter of cell division, results in its conversion to a new species that elutes later than native EGF on a reverse-phase column. This new species, called EGF-X, has only 20% of the biological activity of native EGF. Peptide mapping indicated that the primary structure of EGF-X differs from that of native EGF solely within the first 13 residues. N-Terminal sequencing of EGF-X revealed that about 30% of the polypeptides have been cleaved at the Asp-3/Ser-4 bond. In addition, the yields after the His residue at position 10 were extremely low, indicating that a chemical modification occurs at residue 11 that is incompatible with Edman degradation. We hypothesized that aspartic acid 11 had been converted to an isoaspartyl residue, and this was confirmed with L-isoaspartyl/D-aspartyl methyltransferase, an enzyme that methylates the side-chain carboxyl group of L-isoaspartyl residues but does not recognize normal L-aspartyl residues. EGF-X, but not EGF, was found to be a substrate of this enzyme, and proteolytic digestion of EGF-X with thermolysin localized the site of methylation to a nine-residue peptide containing position 11. We did not observe formation of the isoaspartyl derivative in EGF that had been denatured by reduction of its disulfide bonds. In addition, replacement of the aspartyl residue at position 11 with glutamic acid resulted in a fully active EGF derivative that does not form detectable amounts of EGF-X. We propose that conversion of this aspartyl residue to isoaspartate is a significant nonenzymatic degradation reaction affecting this growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate

Cleavage of specific peptide bonds occurs with aging in the alpha A subunit of bovine alpha-crystallin. One of the breaks occurs at residue Asn-101. This same residue undergoes in vivo deamidation, isomerization, and racemization. Deamidation and isomerization are known to occur via succinimide ring formation of labile asparagine residues. Model studies on peptides have shown that imide formation can also lead to peptide bond cleavage (Geiger, T., and Clarke, S. (1987) J. Biol. Chem. 262, 785-794). In that case, both asparagine and aspartic acid amide would be expected as C termini of the truncated polypeptide, and this is indeed the case in the alpha A-(1-101)-chain. This thus represents a first example of nonenzymatic in vivo peptide bond cleavage in an aging protein through the formation of a succinimide intermediate. In addition, we found that in bovine lens no detectable conversion (through the action of protein-carboxyl methyltransferase) of isoaspartyl to normal aspartyl residues occurs in vivo after deamidation of Asn-101.     

Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase.

Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302-6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-l-isoaspartic acid O-methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 degrees C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR approximately 34 min to approximately 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR approximately 34 min species decreased with a biphasic time-course with t0.5-values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of approximately 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 degrees C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial beta-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32-->Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition.     

Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins

Nonenzymatic intramolecular reactions can result in the deamidation, isomerization, and racemization of protein and peptide asparaginyl and aspartyl residues via succinimide intermediates. To understand the sequence dependence of these reactions, we measured the rate of succinimide formation in a series of synthetic peptides at pH 7.4. These peptides (Val-Tyr-Pro-X-Y-Ala) contained an internal aspartyl, asparaginyl, aspartyl beta-methyl ester, or aspartyl alpha-methyl ester residue (X) followed by a glycyl, seryl, or alanyl residue (Y). The rates of succinimide formation of the asparaginyl peptides were found to be 13.1-35.6 times faster than those of the aspartyl peptides. The rates of succinimide formation for the glycyl peptides were 6.5-17.6 times faster than those of the alanyl peptides, while the rates for the seryl peptides were 1.6-4.5 times faster than those of the alanyl peptides. The overall 232-fold range in these reaction rates for aspartyl and asparaginyl residues suggests that sequence can be an important determinant in their stability in flexible peptides. In proteins, there may be a much larger range in the rates of succinimide formation because specific conformations may greatly enhance or inhibit this reaction.     

Chemical conversion of aspartyl peptides to isoaspartyl peptides. A method for generating new methyl-accepting substrates for the erythrocyte D-aspartyl/L-isoaspartyl protein methyltransferase

Mammalian protein carboxyl methyltransferases have recently been proposed to recognize atypical configurations of aspartic acid and may possibly function in the metabolism of covalently altered cellular proteins. Consistent with this proposal, the tetrapeptide tetragastrin, containing a single "normal" L-aspartyl residue (L-Trp-L-Met-L-Asp-L-Phe-NH2) was found here not to be an in vitro substrate for erythrocyte carboxyl methyltransferase activity. However, chemical treatment of tetragastrin by methyl esterification and then de-esterification of the aspartic acid residue yielded a mixture of peptide products, the major one of which could now be enzymatically methylated. We show here that this new peptide species is the isomeric beta-aspartyl form of tetragastrin (L-iso-tetragastrin; L-Trp-L-Met-L-Asp-L-Phe-NH2), and it appears that isomerization proceeds via an intramolecular succinimide intermediate during the de-esterification procedure. L-iso-Tetragastrin is stoichiometrically methylated (up to 90% in these experiments) with a Km for the enzyme of 5.0 microM. Similar chemical treatment of several other L-aspartyl peptides also resulted in the formation of new methyltransferase substrates. This general method for converting normal aspartyl peptides to isoaspartyl peptides may have application in the reverse process as well.