Advertisement

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

Open AccessPublished:January 15, 1987DOI:https://doi.org/10.1016/S0021-9258(19)75855-4
      This paper is only available as a PDF. To read, Please Download here.
      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.

      REFERENCES

        • Harding J.J.
        Adv. Protein Chem. 1985; 37: 247-334
        • Robinson A.B.
        • Rudd C.J.
        Current Top. Cell. Regul. 1974; 8: 247-295
        • Haley E.E.
        • Corcoran B.J.
        • Dorer F.E.
        • Buchanan D.L.
        Biochemistry. 1966; 5: 3229-3235
        • Bada J.L.
        Methods Enzymol. 1984; 106: 98-115
        • Clarke S.
        Annu. Rev. Biochem. 1985; 54: 479-506
        • Pisano J.J.
        • Prado E.
        • Freedman J.
        Arch. Biochem. Biophys. 1966; 117: 394-399
        • Bornstein P.
        Biochemistry. 1970; 9: 2408-2421
        • Graf L.
        • Bajusz S.
        • Patthy A.
        • Barat E.
        • Cseh G.
        Acta Biochim. Biophys. Acad. Sci. Hung. 1971; 6: 415-418
        • Aswad D.W.
        J. Biol. Chem. 1984; 259: 10714-10721
        • Johnson B.A.
        • Aswad D.W.
        Biochemistry. 1985; 24: 2581-2586
        • McFadden P.N.
        • Clarke S.
        Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2460-2464
        • Bernhard S.A.
        • Berger A.
        • Carter H.H.
        • Katchalski E.
        • Sela M.
        • Shalitin Y.
        J. Am. Chem. Soc. 1962; 84: 2421-2434
        • Ondetti M.A.
        • Deer A.
        • Sheehan J.T.
        • Pluscec J.
        • Kocy O.
        Biochemistry. 1968; 7: 4069-4075
        • Bodanszky M.
        • Kwei J.Z.
        Int. J. Pept. Protein Res. 1978; 12: 69-74
        • Bernhard S.A.
        Ann. N. Y. Acad. Sci. 1983; 421: 28-40
        • Blodgett J.K.
        • Loudon G.M.
        • Collins K.D.
        J. Am. Chem. Soc. 1985; 107: 4305-4313
        • Murray Jr., E.D.
        • Clarke S.
        J. Biol. Chem. 1986; 261: 306-312
        • Murray Jr., E.D.
        • Clarke S.
        J. Biol. Chem. 1984; 259: 10722-10732
        • Perrin D.D.
        • Dempsey B.
        Buffers for pH and Metal Ion Control. Chapman and Hall, London1974: 122
        • Bidlingmeyer B.A.
        • Cohen S.A.
        • Tarvin T.L.
        J. Chromatogr. 1984; 336: 93-104
        • Jones B.N.
        • Gilligan J.P.
        J. Chromatogr. 1983; 266: 471-482
        • Aswad D.W.
        Anal. Biochem. 1984; 137: 405-409
        • McFadden P.N.
        • Clarke S.
        J. Biol. Chem. 1986; 261: 11503-11511
        • Piszkiewicz D.
        • London M.
        • Smith E.L.
        Biochem. Biophys. Res. Commun. 1970; 40: 1173-1178
        • Yuan P.M.
        • Talent J.M.
        • Gracy R.W.
        Mech. Ageing Dev. 1981; 17: 151-162
        • Charache S.
        • Fox J.
        • McCurdy P.
        • Kazazian Jr., H.
        • Winslow R.
        J. Clin. Invest. 1977; 59: 652-658
        • Lewis U.J.
        • Singh R.N.P.
        • Bonewald L.F.
        • Seavey B.K.
        J. Biol. Chem. 1981; 256: 11645-11650
        • Terwilliger T.C.
        • Clarke S.
        J. Biol. Chem. 1981; 256: 3067-3076
        • Janin J.
        • Wodak S.
        • Levitt M.
        • Maigret B.
        J. Mol. Biol. 1978; 125: 357-386
        • Bhat T.N.
        • Sasisekharan V.
        • Vijayan M.
        Int. J. Pept. Protein Res. 1979; 13: 170-184
        • Buchanan D.L.
        • Haley E.E.
        • Markiw R.T.
        Biochemistry. 1962; 1: 612-620
        • Lou M.F.
        Biochemistry. 1975; 14: 3503-3508
        • Tanaka T.
        • Nakajima T.
        J. Biochem. (Tokyo). 1978; 84: 617-625
        • Haley E.E.
        • Corcoran B.J.
        Biochemistry. 1967; 6: 2668-2672
        • Tanaka T.
        • Hirai M.
        • Nakajima T.
        J. Biochem. (Tokyo). 1978; 84: 1147-1153
        • Helfman P.M.
        • Bada J.L.
        Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2891-2894
        • Helfman P.M.
        • Bada J.L.
        Nature. 1976; 262: 279-281
        • Masters P.M.
        • Bada J.L.
        • Zigler Jr., J.S.
        Nature. 1977; 268: 71-73
        • Brunauer L.S.
        • Clarke S.
        J. Biol. Chem. 1986; 261: 12538-12543
        • Clarke S.
        Borchardt R.T. Creveling C.R. Ueland P.M. Biological Methylation and Drug Design. Humana Press, Clifton, NJ1986: 3-14
        • Benoiton N.L.
        The Peptides. 1983; 5: 217-284
        • Srivastava S.K.
        Red Blood Cell and Lens Metabolism. Elsevier Scientific Publishing Co., Inc., New York1980
        • Barber J.R.
        • Clarke S.
        Biochemistry. 1985; 24: 4867-4871
        • McFadden P.N.
        • Clarke S.
        Proc. Natl. Acad. Sci. U. S. A. 1987; 84 (in press)
        • Johnson B.A.
        • Murray Jr., E.D.
        • Clarke S.
        • Glass D.B.
        • Aswad D.W.
        J. Biol. Chem. 1987; 262 (in press)
        • Meinwald Y.C.
        • Stimson E.R.
        • Scheraga H.A.
        Int. J. Peptide Protein Res. 1986; 28: 79-84
        • Yuksel K.U.
        • Gracy R.W.
        Arch. Biochem. Biophys. 1986; 248: 452-459