Perspectives on the Biological Function and Enzymology of Protein Carboxyl Methylation Reactions in Eucaryotic and Procaryotic Cells

  • Steven Clarke
Part of the Advances in Experimental Medicine and Biology book series (NATO ASI F, volume 231)


Enzymes have been detected in all cells examined so far that catalyze the incorporation of methyl groups from S-adenosylmethionine into base-labile linkages on proteins. Although the products of these reactions have many of the properties of methyl esters, their frequent instability has often precluded direct analysis of the linkage chemistry. As a result, previous studies of “protein carboxyl methyltransferase” have included representatives of what has turned out to be at least two distinct classes of enzymes. The often mentioned specificity of such an activity for aspartyl and glutamyl residues is based largely on the apparent chemical reasonableness of such assignments, and specific evidence for the methylation of such residues has rarely been presented. The lack of specific knowledge on the chemistry of such modification reactions has been accompanied by a similar lack of understanding of their physiological role (for reviews of the earlier literature see Gagnon and Heisler, 1979; Borchardt, 1980; Paik and Kim, 1980; O’Dea et al., 1981; Clarke, 1985).


Methylation Reaction Methyl Transferase Asparagine Residue Aspartyl Residue Protein Carboxyl 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ahern, T. J., and Klibanov, A. M., 1985, The mechanism of irreversible enzyme inactivation at 100°C, Science, 228:1280.PubMedCrossRefGoogle Scholar
  2. Ahern, T. J., Casal, J. I., Petsko, G. A., and Klibanov, A. M., 1987, Control of oligomeric enzyme thermostability by protein engineering, Proc. Natl. Acad. Sci. U. S. A., 84:675.PubMedCrossRefGoogle Scholar
  3. Altman, G., 1986, Presence of D-aspartic acid and D-aspartyl peptides in human urine: Implications for protein racemization and turnover, Master’s Thesis, Department of Chemistry and Biochemistry, University of California at Los Angeles.Google Scholar
  4. Aswad, D. W., 1984, 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, J. Biol. Chem., 259:10714.PubMedGoogle Scholar
  5. Aswad, D. W., and Deight, E. A., 1983, Purification and characterization of two distinct isozymes of protein carboxymethylase from bovine brain, J. Neurochem., 40:1718.PubMedCrossRefGoogle Scholar
  6. Aswad, D. W., and Johnson, B. A., 1987, The unusual substrate specificity of eukaryotic protein carboxyl methyltransferases, Trends Biochem. Sci., 12:155.CrossRefGoogle Scholar
  7. Bachmair, A., Finley, D., and Varshavsky, A., 1986, In vivo half-life of a protein is a function of its amino-terminal residue, Science, 234:179.PubMedCrossRefGoogle Scholar
  8. Bada, J. L., 1984, In vivo racemization in mammalian proteins, Methods Enzvmol., 106:98.Google Scholar
  9. Barber, J. R., and Clarke, S., 1983, Membrane protein carboxyl methylation increases with human erythrocyte age: Evidence for an increase in the number of methylatable sites, J. Biol. Chem., 258:1189.PubMedGoogle Scholar
  10. Barber, J. R., and Clarke, S., 1985, Demethylation of protein carboxyl methyl esters: A nonenzymatic process in human erythrocytes?, Biochemistry, 24:4867.PubMedCrossRefGoogle Scholar
  11. Bernhard, S. A., 1983, Nucleophilic displacement reactions at ester and thioester bonds, Annals N. Y. Acad. Sci., 421:28.CrossRefGoogle Scholar
  12. Betz, R., Crabb, J. W., Meyer, H. E., Wittig, R., and Duntze, W., 1987, Amino acid sequences of a-Factor mating peptides from Saccharomyces cerevisiae, J. Biol. Chem., 262:546.PubMedGoogle Scholar
  13. Blodgett, J. K., Loudon, G. M., and Collins, K. D., 1985, Specific cleavage of peptides containing an aspartic acid beta-hydroxamic acid residue, J. Am. Chem. Soc., 197:4305.CrossRefGoogle Scholar
  14. Borchardt, R. T., 1980, S-Adenosyl-L-methionine-dependent macromolecule methyltransferases, J. Med. Chem., 23:347.PubMedCrossRefGoogle Scholar
  15. Bornstein, P., and Balian, G., 1970, The specific nonenzymatic cleavage of bovine ribonuclease with hydroxylamine, J. Biol. Chem., 245:4854.PubMedGoogle Scholar
  16. Brot, N. and Weissbach, H., 1983, Biochemistry and physiological role of methionine sulfoxide residues in proteins, Arch. Biochem. Biophys., 223:271.PubMedCrossRefGoogle Scholar
  17. Brunauer, L. S., and Clarke, S., 1986, Age-dependent accumulation of protein residues which can be hydrolyzed to D-aspartic acid in human erythrocytes” J. Biol. Chem., 261:12538.PubMedGoogle Scholar
  18. Chen, J.-K., and Liss, M., 1978, Evidence of carboxyraethylation of nascent peptide chains on ribosomes, Biochem. Biophys. Res. Commun., 84:261.PubMedCrossRefGoogle Scholar
  19. Chen, Z.-Q., Ulsh, L. S., DuBois, G., and Shin, T. Y., 1985, Post-translational processing of p21 ras proteins involves palmitoylation of the C-terminal tetrapeptide containing cysteine-186, J. Virology, 56:607.PubMedGoogle Scholar
  20. Clarke, S., 1985, Protein carboxyl methyltransferases: Two distinct classes of enzymes, Annu. Rev. Biochem., 54:479.PubMedCrossRefGoogle Scholar
  21. Clarke, S., 1987, Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins, submitted for publication.Google Scholar
  22. Clarke, S., McFadden, P. N., O’Connor, C. M., and Lou, L. L., 1984, Isolation of D-aspartic acid beta-methyl ester from erythrocyte carboxyl methylated proteins, Methods Enzvmol., 106:331.Google Scholar
  23. Clarke, S., Panasenko S., Sparrow, K., and Koshland, D. E. Jr., 1980, In vitro methylation of bacterial Chemotaxis proteins: Characterization of protein methyltransferase activity in crude extracts of Salmonella typhimurium, J. Supramol. Struct., 13:315.PubMedCrossRefGoogle Scholar
  24. Di Donato, A., Galletti, P., and D’Alessio, G., 1986, Selective deamidation and enzymatic methylation of seminal ribonuclease, Biochemistry, 25:8361.PubMedCrossRefGoogle Scholar
  25. Diliberto, E. J., Jr., and Axelrod, J., 1976, Regional and subcellular distribution of carboxymethylase in brain and other tissues, J. Neurochem., 26:1159.PubMedCrossRefGoogle Scholar
  26. Flatmark, T., 1966, On the heterogeneity of beef heart cytochrome c: III. A kinetic study of the non-enzymatic deamidation of the main subfractions (Cy I — Cy III), Acta Chem. Scand., 20:1487.PubMedCrossRefGoogle Scholar
  27. Freitag, C. and Clarke S., 1981, Reversible methylation of cytoskeletal and membrane proteins in intact human erythrocytes, J. Biol. Chem., 256:6102.PubMedGoogle Scholar
  28. Gagnon, C., and Heisler, S., 1979, Protein carboxyl-methylation: Role in exocytosis and Chemotaxis, Life Sci., 25:993.PubMedCrossRefGoogle Scholar
  29. Galletti, P., Ingrosso, D., Iardino, P., Manna, C., Pontoni, G., and Zappia, V., 1986, Enzymatic basis for the calcium-induced decrease of membrane protein methyl esterification in intact erythrocytes: Evidence for an impairment of S-adenosylmethionine synthesis, Eur. J. Biochem.. 154:489.PubMedCrossRefGoogle Scholar
  30. Galletti, P., Ingrosso, D., Nappi, A., Gragnaniello, V., Iolascon, A., Pinto, L., 1983, Increased methyl esterification of membrane proteins in aged red blood cells: Preferential esterification of ankyrin and Band 4.1 cytoskeletal proteins, Eur. J. Biochem., 135:25.PubMedCrossRefGoogle Scholar
  31. Geiger, T. and Clarke, S., 1987, Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides: Succinimide-linked reactions that contribute to protein degradation, J. Biol. Chem., 262:785.PubMedGoogle Scholar
  32. Ishibashi, Y., Sakagami, Y., Isogai, A., and Suzuki, XT, 1984, Tremergogens A-9291-I and A-9291-VIII: Peptidyl sex hormones of Tremella brasiliensis, Biochemistry, 23:1399.CrossRefGoogle Scholar
  33. Johnson, B. A., and Aswad, D. W., 1985, Enzymatic protein carboxyl methylation at physiological pH: Cyclic imide formation explains rapid methyl turnover, Biochemistry, 24:2581.PubMedCrossRefGoogle Scholar
  34. Johnson, B. A., Freitag, N. E., and Aswad, D. W., 1985, Protein carboxyl methyltransferase selectively modifies an atypical form of calmodulin: Evidence for methylation at deamidated asparagine residues, J. Biol. Chem., 260:10913.PubMedGoogle Scholar
  35. Johnson, B. A., Murray, E. D., Jr., Clarke, S., Glass, D. B., and Aswad, D. W., 1987a, Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides, J. Biol. Chem., 262:5622.PubMedGoogle Scholar
  36. Johnson, B. A., Langmack, E. L., and Aswad, D. W., 1987b, Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase, J. Biol. Chem., 262:in press.Google Scholar
  37. Kleene, S. J., Hobson, A. C., and Adler, J., 1979, Isolation of glutamic acid methyl ester from an Escherichia coli membrane protein involved in Chemotaxis, J. Biol. Chem., 252:3214.Google Scholar
  38. Lewis, U. J., Singh, R. N. P., Bonewald, L. F., and Seavey, B. K., 1981, Altered proteolytic cleavage of human growth hormone as a result of deamidation, J. Biol. Chem., 256:11645.PubMedGoogle Scholar
  39. Lou, L. L. and Clarke, S., 1987, Enzymatic methylation of Band 3 anion transporter in intact human erythrocytes, Biochemistry, 26:52.PubMedCrossRefGoogle Scholar
  40. Lowenson, J., and Clarke, S., 1987, Protein carboxyl methyltransferase from human erythrocytes: Substrate specificity with L-isoaspartyl and D-aspartyl-containing peptides and proteins, Fed. Proc., in press.Google Scholar
  41. McFadden, P. N., and Clarke, S., 1982, Methylation at D-aspartyl residues in red cells: A possible step in the repair of aged membrane proteins, Proc. Natl. Acad. Sci. U. S. A., 79:2460.PubMedCrossRefGoogle Scholar
  42. McFadden, P. N., and Clarke, S., 1986, 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, J. Biol. Chem., 261:11503.PubMedGoogle Scholar
  43. McFadden, P. N., and Clarke, S., 1987, Conversion of isoaspartyl peptides to normal peptides: Implications for the cellular repair of aged membrane proteins, Proc. Natl. Acad. Sci. U. S. A., 84:2595.PubMedCrossRefGoogle Scholar
  44. Midelfort, C. F., and Mehler, A. H., 1972, Deamidation in vivo of an asparagine residue of rabbit muscle aldolase, J. Biol. Chem., 247:3618.PubMedGoogle Scholar
  45. Momand, J., and Clarke, S., 1987, Rapid degradation of D- and L-succinimide peptides by a post-proline endopeptidase in human erythrocytes, submitted for publication.Google Scholar
  46. Moo-Penn, W., Jue, D. L., Bechtel, K. C., Johnson, M. H., Schmidt, R. M., McCurdy, P. R., Fox, J., Bonaventura, J., Sullivan, B., and Bonaventura, C., 1976, Hemoglobin Providence: A human hemoglobin variant occurring in two forms in vivo, J. Biol. Chem., 251:7557.PubMedGoogle Scholar
  47. Murray, E. D., Jr., and Clarke, S., 1984, Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase: Detection of a new site of methylation at isomerized L-aspartyl residues, J. Biol. Chem., 259:10722.PubMedGoogle Scholar
  48. Murray, E. D., Jr., and Clarke, S., 1986, Metabolism of a synthetic L-isoaspartyl-containing hexapeptide in erythrocyte extracts: Enzymatic methyl esterification is followed by nonenzymatic succinimide formation, J. Biol. Chem., 261:310.Google Scholar
  49. Nemethy, G., and Scheraga, H. A., 1977, Protein folding, Quart. Rev. Biophys., 10:239.CrossRefGoogle Scholar
  50. Nowlin, D. M., Nettleton, D. O., Ordal, G. W., and Hazelbauer, G. L., 1985, Chemotactic transducer proteins of Escherichia coli exhibit homology with methyl-accepting proteins from distantly related bacteria, J. Bacteriol., 163:262.PubMedGoogle Scholar
  51. O’Connor, C. M., 1987, Regulation and subcellular distribution of a protein methyltransferase and its damaged aspartyl substrate sites in developing Xenopus oocytes, J. Biol. Chem., 262:in press.Google Scholar
  52. O’Connor, C. M., Aswad, D. W., and Clarke, S., 1984, Mammalian brain and erythrocyte carboxyl methyltransferases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl residues in structurally altered protein substrates, Proc. Natl. Acad. Sci. U. S. A., 81:7757.PubMedCrossRefGoogle Scholar
  53. O’Connor, C. M., and Clarke, S., 1983, Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro: Saturation of intracellular sites in vivo, J. Biol. Chem., 258:8485.PubMedGoogle Scholar
  54. O’Connor, C. M., and Clarke, S., 1984, Carboxyl methylation of cytosolic proteins in intact human erythrocytes: Identification of numerous methyl-accepting proteins including hemoglobin and carbonic anhydrase, J. Biol. Chem., 259:2570.PubMedGoogle Scholar
  55. O’Connor, C. M., and Clarke, S., 1985a, Specific recognition of altered polypeptides by widely distributed methyltransferases, Biochem. Biophys. Res. Commun., 132:1144.PubMedCrossRefGoogle Scholar
  56. O’Connor, C. M., and Clarke, S., 1985b, Analysis of erythrocyte protein methyl esters by two-dimensional gel electrophoresis under acidic separating conditions, Anal. Biochem., 148:79.PubMedCrossRefGoogle Scholar
  57. O’Dea, R. F., Viveros, O. H., Diliberto, E. J. Jr., 1981, Protein carboxymethylation — Role in the regulation of cell functions, Biochem. Pharmacol., 30:1163.PubMedCrossRefGoogle Scholar
  58. Ota, I. M., Ding, L., and Clarke, S., 1987, Methylation at specific altered aspartyl and asparaginyl residues in glucagon by the erythrocyte protein carboxyl methyltransferase, J. Biol. Chem., 262:in press.Google Scholar
  59. Paik, W. K., and Kim S., 1980, “Protein Methylation,” Wiley, New York, pp. 202–231.Google Scholar
  60. Sibanda, B. L., and Thornton, J. M., 1985, Beta-Hairpin families in globular proteins, Nature, 316:74.CrossRefGoogle Scholar
  61. Stock, J. and Simms, S., 1987, Methylation, demethylation, and dearaidation at glutamate residues in membrane chemoreceptor proteins, this volume.Google Scholar
  62. Stock, J. and Stock, A., 1987, What is the role of receptor methylation in bacterial Chemotaxis?, Trends Biochem. Sci., 12:in press.Google Scholar
  63. Svasti, J., and Milstein, C., 1972, The complete amino acid sequence of a mouse kappa light chain, Biochem. J., 128:427.PubMedGoogle Scholar
  64. Terwilliger, T. C., and Clarke, S., 1981, Methylation of membrane proteins in human erythrocytes: Identification and characterization of polypeptides methylated in lysed cells, J. Biol. Chem., 256:3067.PubMedGoogle Scholar
  65. Van der Werf, P., and Koshland, D. E. Jr., 1977, Identification of a gamma-glutamyl methyl ester in bacterial membrane protein involved in Chemotaxis, J. Biol. Chem., 252:2793.Google Scholar
  66. Voorter, C. E. M., Mulders, J. W. M., Bloemendal, H., and de Jong, W. W., 1987, Phosphorylation and deamidation of the eye lens protein alpha-crystallin, this volume.Google Scholar
  67. Wilson, J. M., Landa, L. E., Kobayashi, R, and Kelley, W. N., 1982, Human hypoxanthine-guanine phosphoribosyltransferase: Post-translational modification of the erythrocyte enzyme, J. Biol. Chem., 257:14830.PubMedGoogle Scholar
  68. Yuan, P. M., Talent, J. M., and Gracy, R. W., 1981, Molecular basis for the accumulation of acidic isozymes of triosephosphate isomerase on aging, Mechan. Ageing Develop., 17:151.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1988

Authors and Affiliations

  • Steven Clarke
    • 1
  1. 1.Department of Chemistry & Biochemistry and the Molecular Biology InstituteUCLALos AngelesUSA

Personalised recommendations