Stoichiometric methylation of porcine adrenocorticotropin by protein carboxyl methyltransferase requires deamidation of asparagine 25. Evidence for methylation at the alpha-carboxyl group of atypical L-isoaspartyl residues

The enzymatic methylation of porcine adrenocorticotropin (ACTH) in both its native form and a form which is deamidated at asparagine 25 has been compared using purified protein carboxyl methyltransferase from bovine brain. Incubation of deamidated ACTH with high concentrations of methyltransferase resulted in near stoichiometric levels of methyl incorporation (78 mol %), while the methylation of native ACTH was highly substoichiometric (3-12 mol %). The Km and Vmax for deamidated ACTH were 1.9 microM and 11,200 pmol/min/mg, respectively, making this peptide the most specific substrate known for the mammalian methyltransferase. Deamidation of asparagine 25 leads to the formation of an atypical isopeptide bond in which the resulting aspartyl residue is linked to the adjacent glycine 26 via its side-chain beta-carboxyl group rather than the usual alpha-carboxyl linkage (Gráf, L., Bajusz, S., Patthy A., Barát, E., and Cseh, G. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 415-418; Bornstein, P., and Balian, G. (1977) Methods Enzymol. 47, 132-145). A synthetic isopeptide (beta-linked) analog of deamidated ACTH serves as a highly effective substrate for the methyltransferase, but the corresponding normal (alpha-linked) peptide does not, indicating that this enzyme selectively recognizes the alpha-carboxyl group of atypical beta-linked L-aspartyl residues (see also accompanying paper (Murray, E.D., Jr., and Clarke, S. (1984) J. Biol. Chem. 259, 10722-10732]. Methylation of atypical beta-linked L-aspartyl residues resulting from deamidation can account for previous observations that in vitro protein carboxyl methylation in mammalian systems almost always occurs with a low stoichiometry and that these protein methyl esters are considerably less stable than most chemically formed protein methyl esters.     

Metabolism of a synthetic L-isoaspartyl-containing hexapeptide in erythrocyte extracts. Enzymatic methyl esterification is followed by nonenzymatic succinimide formation

The synthetic peptide, L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala, is a substrate for the erythrocyte and brain protein carboxyl methyltransferases. These enzymes catalyze the methyl esterification of the free alpha-carboxyl group of the isoaspartyl residue, to which the glycyl residue is linked through the side chain beta-carboxyl group. In this work, we show that the alpha-methyl ester of this peptide was rapidly demethylated (t1/2 = 4 min at 37 degrees C, pH 7.4) in erythrocyte cytosolic extracts and that the product of this reaction appears to be the succinimide ring derivative of the peptide. The rate of demethylation, measured at either pH 6.0 or 7.4, was the same in buffer and erythrocyte extracts, suggesting that succinimide formation was a nonenzymatic reaction. The L-succinimide is more stable than the ester, but can be hydrolyzed in buffer at pH 7.4 (t1/2 = 180 min at 37 degrees C) to give a mixture of about 75% isoaspartyl peptide and 25% normal aspartyl peptide. The metabolism of the succinimide hexapeptide in erythrocyte extracts appears to be more complex, however. The implications of this work for the methylation and demethylation of cellular proteins containing structurally altered aspartyl residues are discussed.     

Endogenous Substrates for Protein Carboxyl Methyltransferase in Cytosolic Fractions of Bovine Brain

Abstract: A method of polyacrylamide gel electrophoresis utilizing the discontinuous pH-stacking gel format, the cationic detergent cetylpyridinium chloride, and an acidic buffer system has been applied to detection of specific substrates for protein carboxyl methyltransferase (PCM, EC 2.1.1.24) in cytosol fractions of bovine cerebral cortex. This electrophoresis system produces a high-resolution separation of proteins while preventing spontaneous hydrolysis of protein carboxyl methyl esters. Separation occurs largely on the basis of molecular weight. By running polyacrylamide gels at 4°C or 25°C, it was possible to demonstrate that any specific methyl-accepting protein is modified to form a labile methyl ester rather than the more stable N -derivative. Using this system, we have found that partially purified fractions of PCM contain a variety of endogenous methyl-accepting proteins. The apparent specificity of these substrates varies widely; some apparently abundant proteins show little or no methylation, while other apparently less abundant proteins exhibit a relatively high degree of methylation. One protein, with an apparent M_r of 46,000, exhibited an exceptional degree of methylation. Two distinct classes of protein carboxyl methyl esters could be distinguished by their differing susceptibility to nonenzymatic hydrolysis. The possible relevance of our findings to the recent suggestion that PCM specifically methylates abnormal d-aspartyl residues in age-racemized proteins is considered.     

Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides

Prolonged incubation of L-isoaspartate-containing forms of lactate dehydrogenase (231-242), sperm activating peptide, and adrenocorticotropin (22-27) at 37 degrees C, pH 7.4, with S-adenosyl-L-methionine and protein carboxyl methyltransferase from bovine brain leads to extensive conversion of the atypical isopeptide bond to a normal peptide bond. For the lactate dehydrogenase-related peptide, conversion was 80% complete after 24 h. For the other two peptides, conversion reached a level of approximately 65% after 48 h. The mechanism of conversion involves (i) rapid enzymatic methylation of the alpha-carboxyl of the L-iso-Asp residue; (ii) nonenzymatic demethylation resulting in formation of an L-aspartyl cyclic imide; and (iii) a slow, nonenzymatic hydrolysis of the cyclic imide to form a mixture of 15-25% normal L-Asp peptide and 75-85% L-iso-Asp peptide. The regenerated L-iso-Asp peptide is remethylated and the cycle is repeated. The extent of conversion is limited by a competing side reaction wherein the L-imide slowly racemizes, leading to the formation of mainly D-iso-Asp peptide, which is not a substrate for the methyltransferase. The ability of protein carboxyl methyltransferase to initiate conversion of L-iso-Asp residues to normal L-Asp suggests a possible role for this enzyme in facilitating the repair or degradation of deamidated proteins in vivo.     

A new type of protein methylation activated by tyrphostin A25 and vanadate

It has been reported that S-adenosylmethionine-dependent protein methylation in rat kidney extracts can be greatly stimulated by tyrphostin A25, a tyrosine kinase inhibitor. We have investigated the nature of this stimulation. We find that addition of tyrphostin A25, in combination with the protein phosphatase inhibitor vanadate, leads to the stimulation of methylation of polypeptides of 64, 42, 40, 36, 31, and 15 kDa in cytosolic extracts of mouse kidney. The effect of tyrphostin appears to be relatively specific for the A25 species. The enhanced methylation does not represent the activity of the families of protein histidine, lysine or arginine methyltransferases, nor that of the l-isoaspartyl/d-aspartyl methyltransferase, enzymes responsible for the bulk of protein methylation in most cell types. Chemical and enzymatic analyses of the methylated polypeptides suggest that the methyl group is in an ester linkage to the protein. In heart extracts, we find a similar situation but here the stimulation of methylation is not dependent upon vanadate and an additional 18 kDa methylated species is found. In contrast, little or no stimulation of methylation is found in brain or testis extracts. This work provides evidence for a novel type of protein carboxyl methylation reaction that may play a role in signaling reactions in certain mammalian tissues.     

Kinetic and electrophoretic analysis of transmethylation reactions in intact Xenopus laevis oocytes

Transmethylation reactions in fully grown Xenopus oocytes were analyzed following the microinjection of S-adenosyl-L-[methyl-3H]methionine (AdoMet). The size of the endogenous AdoMet pool, measured by cation exchange high pressure liquid chromatography is 5.91 pmol/oocyte. The AdoMet pool turns over with a half-time of 2 h, at a rate of 2.07 pmol/h/oocyte. Fractionation experiments indicate that approximately one-third of the AdoMet in oocytes is utilized for protein carboxylmethylation reactions and another third is metabolized into small molecules which are secreted. The remainder of the intracellular AdoMet is used primarily for protein N-methylation reactions, although some methylation of phospholipids and nucleic acids also occurs. Polyacrylamide gel electrophoresis of 3H-methylated proteins at pH 2.4 in the presence of sodium dodecyl sulfate demonstrated that methyl esters are associated with a heterogeneous group of proteins in both the nucleus and cytoplasm of oocytes, coincident with the subcellular distribution of the protein D-aspartyl, L-isoaspartyl methyl transferase (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). The protein methyl esters associated with oocyte proteins turn over rapidly, as evidenced from the presence of [3H]methanol in the medium. The calculated rate of protein carboxyl methylation, 0.7 pmol/h/oocyte, is similar to that of protein synthesis in oocytes, suggesting that the modification of derivatized aspartyl residues represents a major pathway in oocyte protein metabolism. Since the formation of protein methyl esters is unaffected by cycloheximide, it is unlikely that methyl-accepting sites on oocyte proteins arise primarily from errors in protein synthesis. Unlike protein carboxyl methylation reactions, protein N-methylation reactions are closely linked to protein synthesis, and the methyl group linkages are stable over a period of at least 4 h. Numerous protein acceptors for N-methylation reactions were identified by polyacrylamide gel electrophoresis.     

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.     

Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.

A strategy that facilitates the identification of substrates for protein carboxyl methyltransferases that form "stable" methyl esters, i.e., those that remain largely intact during conventional polyacrylamide gel electrophoresis is described. Rat PC12 cells were cultured in the presence of adenosine dialdehyde (a methylation inhibitor) to promote the accumulation of hypomethylated proteins. Nonidet P-40 cell extracts were then incubated in the presence of S-[methyl-3H]adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. After labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel slices were incubated in 4 N methanesulfonic acid or 6 N HCl to hydrolyze methyl esters. The resulting [3H]methanol was detected by trapping in liquid scintillation fluid. Seven carboxyl methylated proteins were observed with masses ranging from 18 to 96 kDa. Detection of five of these proteins required prior treatment of cells with adenosine dialdehyde, while methyl incorporation into one protein at 18 kDa was substantially enhanced by the treatment. The use of acidic conditions for methyl ester hydrolysis has an important advantage over assays that utilize alkaline hydrolysis conditions. In PC12 cells, and possibly other cell types where there are significant levels of arginine methylation, the methanol signal becomes obscured by high levels of volatile methylamines generated under the alkaline conditions. Carrying out diffusion assays under acidic conditions eliminates this interference. Adenosine dialdehyde, by virtue of increasing the methyl-accepting capacity of substrates for protein carboxyl methyltransferases, in combination with a more selective assay for carboxyl methylation, should prove useful in the isolation and characterization of new protein carboxyl methyltransferases and their substrates.     

Guanine nucleotide-dependent carboxyl methylation of mammalian membrane proteins

A guanine nucleotide-dependent protein carboxyl methylation is demonstrated in mammalian cell membranes. The methylation of membrane proteins of Mr 20,000-23,000 requires S-adenosylmethionine, GTP or nonhydrolyzable GTP analogs, and a cytoplasmic methyltransferase. The protein methyl groups are stable at neutral pH and under basic conditions hydrolyze to produce methanol. The specific methyl acceptor proteins and methyltransferases varied between tissues and cell types, suggesting that these methylations have cell-specific functions. The guanine nucleotide-dependent carboxyl methylations provide a possible mechanism for regulating the function of GTP-binding membrane proteins in the transduction of receptor-mediated signals of eukaryotic cells.     

Pitfalls in the measurement of protein carboxyl methylation during chemotaxis of Dictyostelium discoideum

In Dictyostelium discoideum, a chemoacttractant-stimulated incorporation of radioactivity from [methyl-³H]methionine into protein in the presence of cycloheximide has previously been assumed to represent carboxyl methylation. In this paper, however, evidence is presented which demonstrates it to be a non-covalent binding of methionine to structural components of the cell. Certain pitfalls in the assay of carboxyl methylation by measurement of methanol production are described, and the assay has been optimized to avoid measurement of other volatile compounds. When carboxyl methylation is measured as the amount of methanol formed from methylated protein, methanol production is near the limit of detection in all current methods of assay. No increase of methanol production could be observed upon a single or repeated stimulation of aggregative cells with their chemoattractant, cyclic AMP. We conclude that carboxyl methylation is either absent in Dictyostelium, or that it involves only a small amount of specific methyl acceptors.     

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.     

Studies in fluorescence histochemistry. VII. The mechanism of the complex reactions that may take place between protein carboxyl groups and hot mixtures of acetic anhydride and pyridine in the acetic anhydride-salicylhydrazide-zinc (or fluorescent ketone) method for localizing protein C-terminal carboxyl groups

Synopsis Theoretical arguments are marshalled with experimental evidence to support claims made previously (Stoward & Burns, 1967, 1968) that at 60°C acetic anhydride in the presence of pyridine transforms C-terminal carboxyl groups of proteins to methyl ketones and converts side-chain carboxyl groups to acid anhydrides. On balance the experimental evidence also supports another claim, namely that the methyl ketones thus formed from C-terminal carboxyl groups may be demonstrated specificallyin situ by the intense fluorescence they emit after treatment successively with aqueous solutions of salicylhydrazide and zinc acetate.The experiments carried out included ones favouring the exclusive formation of acid anhydrides, blocking of possible anhydrides with aromatic amines or alcohols, hydrolysis of anhydrides with alkalis, and prior methylation of carboxyl groups.     

Formation of cyclic imide-like structures upon the treatment of calmodulin and a calmodulin peptide with heat

Protein cyclic imide is the putative intermediate in the formation of sites of carboxyl-methylation in eukaryotic proteins. Conditions known to induce the formation of a cyclic imide in model peptides have been applied to a protein, calmodulin. Heating of calmodulin in the dry state at 100 degrees C for 24 h after lyophilization from a pH 2.0 or pH 6.0 solution produces derivatives with altered chromatographic properties in anion-exchange HPLC. At pH 6.0, complete activity of calmodulin was retained. Analysis with Fourier transform infrared (FTIR)-photoacoustic spectroscopy demonstrated the presence of a new structure in the calmodulin molecule consistent with modification of carboxylic acid groups. The conversion of calmodulin is dependent upon the absence of Ca2+ (the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid). A peptide analogous to the calcium binding regions of calmodulin, Asp-Lys-Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys-Glu, is also converted, upon heating, to chromatographically different forms in reversed-phase chromatography. This process is also dependent upon the absence of calcium. Sequence analysis of the peptide derivatives reveals a second amino terminus, implicating peptide bond hydrolysis in the product. A dipeptide, Asp-Gly, known to form a cyclic imide structure under similar conditions is also hydrolyzed during sequence analysis consistent with cleavage occurring at the position of the cyclic imide structure. Asp3 is suggested to be the site of cyclic imide formation in the calmodulin peptide. The presence of a cyclic imide structure is also confirmed by the application of FTIR-photoacoustic spectroscopy. These data suggest that cyclic imide formation in calmodulin has been induced, possibly at one, or more, of the calcium binding loops of the protein. These modification reactions may provide a basis for future investigations of cyclic imide formation in proteins.     

A novel post-translational modification of yeast elongation factor 1A. Methylesterification at the C terminus

Protein methylation reactions can play important roles in cell physiology. After labeling intact Saccharomyces cerevisiae cells with S-adenosyl-l-[methyl-(3)H]methionine, we identified a major methylated 49-kDa polypeptide containing [(3)H]methyl groups in two distinct types of linkages. Peptide sequence analysis of the purified methylated protein revealed that it is eukaryotic elongation factor 1A (eEF1A, formerly EF-1alpha), the protein that forms a complex with GTP and aminoacyl-tRNAs for binding to the ribosomal A site during protein translation. Previous studies have shown that eEF1A is methylated on several internal lysine residues to give mono-, di-, and tri-N-epsilon-methyl-lysine derivatives. We confirm this finding but also detect methylation that is released as volatile methyl groups after base hydrolysis, characteristic of ester linkages. In cycloheximide-treated cells, methyl esterified eEF1A was detected largely in the ribosome and polysome fractions; little or no methylated protein was found in the soluble fraction. Because the base-labile, volatile [methyl-(3)H]radioactivity of eEF1A could be released by trypsin treatment but not by carboxypeptidase Y or chymotrypsin treatment, we suggest that the methyl ester is present on the alpha-carboxyl group of its C-terminal lysine residue. From the results of pulse-chase experiments using radiolabeled intact yeast cells, we find that the N-methylated lysine residues of eEF1A are stable over 4 h, whereas the eEF1A carboxyl methyl ester has a half-life of less than 10 min. The rapid turnover of the methyl ester suggests that the methylation/demethylation of eEF1A at the C-terminal carboxyl group may represent a novel mode of regulation of the activity of this protein in yeast.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

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.     

Isosteric conversion of protein carboxyl groups into carboxamide groups. II. Application to lysozyme

For isosteric conversion of carboxyl groups of proteins into amide groups, ammonolysis of protein esters under mild conditions was attempted. Ammonolysis of methyl esters of lysozyme and bovine serum albumin proved to be incomplete. Highly reactive N-ethylsalicylamide esters of guanylated lysozyme were therefore prepared by subjecting the protein to reaction with N-ethylbenzisoxazolium ion at pH 4.2, 0 degree. Per molecule, 5-7 ester groups were introduced, with concomitant decrease of activity of 80-90%. Only 0.3 tyrosine was modified. On hydrolysis at pH 9.2 the activity was completely restored. At pH 7.9 three classes of ester groups could be distinguished: one group of high rate of hydrolysis (k1 = 1.5 min-1), three groups of intermediate rate (k2 = 0.13 min-1) and two groups of low rate (k3 = 0.018 min-1). The intermediate rate approximated the rate of hydrolysis of the model compound benzoylglycine N-ethylsalicylamide ester (k = 0.15 min-1). Ammonolysis at pH 9.2 in 2.0 M ammonia/ammonium acetate provided complete conversion of the ester groups into amide groups without restoration of activity, confirming the essentiality of certain carboxyl groups. In particular, rearrangement of the ester groups into relatively stable imide groups by O-N acyl migration was found to be completely absent. When native lysozyme was esterified with N-ethylbenzisoxazolium ion the activity did not completely return on hydrolysis.     

Methylation of microinjected isoaspartyl peptides in Xenopus oocytes. Competition with protein carboxyl methylation reactions

Xenopus oocytes possess a highly conserved protein carboxyl methyltransferase postulated to function in the repair or metabolism of age-damaged protein aspartyl residues (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). Three hexapeptides of the general sequence Val-Tyr-Pro-isoAsp-X-Ala, in which isoAsp represents an L-isoaspartyl residue and X represents Gly, Ser, or Ala, are methylated with the same order of preference following their microinjection into oocytes as in a purified system containing bovine brain protein carboxyl methyltransferase and S-adenosyl-L-[methyl-3H]methionine. The affinities of the enzyme for the glycyl, seryl, and alanyl variants of the peptides in vitro are 4.25, 3.04, and 1.67 microM, respectively. A nonapeptide of the sequence Lys-Ala-Ser-Ala-isoAsp-Leu-Ala-Lys-Tyr is a higher affinity substrate for the methyltransferase in vitro, characterized by a Km of 0.88 microM, but it is modified to a lesser extent in oocytes, partially because of its reduced stability in cytoplasm. The hexapeptide Val-Tyr-Pro-Asp-Gly-Ala, which contains an aspartyl residue in the usual stereoconfiguration, is not methylated either in vitro or in intact oocytes. Microinjection of any of the four isoaspartyl-containing peptides greatly stimulates total carboxyl methylation in oocytes, with rate increases ranging from 19- to 51-fold after the injection of 30 pmol of peptide. The protein ovalbumin is also modified following its microinjection into oocytes to near its calculated methyl-accepting capacity. Each of the isoaspartyl peptides can act as a competitive inhibitor of ovalbumin methylation both in vitro and in microinjected oocytes. The inhibitory potencies of the peptides parallel their specific methyl-accepting activities. The results demonstrate that the oocyte may be a useful model for studying the significance of protein carboxyl methylation because of the large functional excess of methylation capacity and the fidelity of the reactions compared to those observed in purified systems. This excess capability may have physiological significance when structurally abnormal proteins accumulate as a result of cellular stress and or aging.     

Inhibition of protein carboxyl methylation by S-adenosyl-L-homocysteine in intact erythrocytes. Physiological consequences

S-Adenosyl-L-homocysteine was used to inhibit the methylation of carboxylic acid residues of membrane proteins in intact human erythrocytes. Incubation of erythrocytes for 24 h with 5 mM each of adenosine and L-homocysteine resulted in the intracellular accumulation of S-adenosyl-L-homocysteine and substantially inhibited membrane protein carboxyl methylation. From the degree of inhibition and from the observed turnover of methylated proteins, we estimate that the number of protein methyl esters in cells incubated with adenosine and L-homocysteine for 20 h is less than 20% that of cells incubated without these inhibitors. No significant differences in the physical deformability properties of the membrane of these hypomethylated cells were detected. However, there was a small but significant (p less than 0.001) increase in the amount of membrane protein D-aspartyl residues in these cells compared to control cells. These observations are consistent with the hypothesis that methylation of membrane proteins at D-aspartyl residues may result in the selective removal or repair of these uncommon residues.     

Methylation at specific altered aspartyl and asparaginyl residues in glucagon by the erythrocyte protein carboxyl methyltransferase

Protein carboxyl methyltransferases from erythrocytes and brain appear to catalyze the esterification of L-isoaspartyl and/or D-aspartyl residues but not of normal L-aspartyl residues. In order to identify the origin of these unusual residues which occur in subpopulations of a variety of cellular proteins, we studied the in vitro methylation by the erythrocyte enzyme of glucagon, a peptide hormone of 29 amino acids containing 3 aspartyl residues and a single asparagine residue. Methylated glucagon was digested with either trypsin, chymotrypsin, pepsin, or endoproteinase Arg C, and the labeled fragments were separated by high-performance liquid chromatography and identified. In separate experiments, methyl acceptor sites were determined by digesting glucagon first with proteases and then assaying purified glucagon fragments for methyl acceptor activity. Using both approaches, we found that the major site of methylation, accounting for about 62% of the total, was at the position of Asp-9. Chemical analysis of fragments containing this residue indicated that this site represents an L-isoaspartyl residue. A second site of methylation, representing about 23% of the total, was detected at the position of Asn-28 and was also shown to represent an L-isoaspartyl residue. Methyl acceptor sites were not detected at the positions of Asp-15 or Asp-21. Preincubation of glucagon under basic conditions (0.1 M NH4OH, 3 h, 37 degrees C) increased methylation at the Asn-28 site by 4-8-fold while methylation at the Asp-9 site remained unchanged. These results suggest that methylation sites can originate from both aspartyl and asparaginyl residues and that these sites may be distinguished by the effect of base treatment.