Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of leu129 or asp119

Tadashi Ueda, Hidenori Yamada, Taiji Imoto

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

In the cross-linking reaction of lysozyme between Leu129 (α-COO-) and Lys13 (ε-NH3 + using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a side reaction of the peptide bond inversion from α to β between A and Gly102 was greatly reduced by addition of β-(1,4)-linked trimer of N-acetyl-D-glucosamine [(NAG)3] When methylamine or 2-hydroxyethylamine was further added, the extent of the cross-link formation was decreased and the derivative where the α-carboxyl group of Leu129 was modified with the amine was newly obtained. On the other hand, when ammonia was added, the β-carboxyl group of Asp119 instead of the α-carboxyl group was mainly amidated. From these results, the presence of a salt bridge between Asp119 and Arg125 besides that between Lys13 and Leu129 is proposed. Enzymatic activities of the derivatives prepared here indicated that the modification of the α-carboxyl group reduced the activity to ̃ 90% of that of native lysozyme. Des-Leu129 lysozyme, which lacks Leu129 also showed ̃ 90% of the activity of native lysozyme. Therefore, the salt bridge between Lys13 and Leu129 may play some role in maintaining the active conformation of lysozyine.

Original languageEnglish
Pages (from-to)189-193
Number of pages5
JournalProtein Engineering, Design and Selection
Volume1
Issue number3
DOIs
Publication statusPublished - Jun 1 1987

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Carbodiimides
Muramidase
Enzymes
Salts
Glucosamine
Derivatives
Acetylglucosamine
Cross Reactions
Ammonia
Peptides
Amines
Conformations

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Molecular Biology
  • Bioengineering
  • Biotechnology
  • Biochemistry

Cite this

Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of leu129 or asp119 . / Ueda, Tadashi; Yamada, Hidenori; Imoto, Taiji.

In: Protein Engineering, Design and Selection, Vol. 1, No. 3, 01.06.1987, p. 189-193.

Research output: Contribution to journalArticle

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abstract = "In the cross-linking reaction of lysozyme between Leu129 (α-COO-) and Lys13 (ε-NH3 + using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a side reaction of the peptide bond inversion from α to β between A and Gly102 was greatly reduced by addition of β-(1,4)-linked trimer of N-acetyl-D-glucosamine [(NAG)3] When methylamine or 2-hydroxyethylamine was further added, the extent of the cross-link formation was decreased and the derivative where the α-carboxyl group of Leu129 was modified with the amine was newly obtained. On the other hand, when ammonia was added, the β-carboxyl group of Asp119 instead of the α-carboxyl group was mainly amidated. From these results, the presence of a salt bridge between Asp119 and Arg125 besides that between Lys13 and Leu129 is proposed. Enzymatic activities of the derivatives prepared here indicated that the modification of the α-carboxyl group reduced the activity to ̃ 90{\%} of that of native lysozyme. Des-Leu129 lysozyme, which lacks Leu129 also showed ̃ 90{\%} of the activity of native lysozyme. Therefore, the salt bridge between Lys13 and Leu129 may play some role in maintaining the active conformation of lysozyine.",
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N2 - In the cross-linking reaction of lysozyme between Leu129 (α-COO-) and Lys13 (ε-NH3 + using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a side reaction of the peptide bond inversion from α to β between A and Gly102 was greatly reduced by addition of β-(1,4)-linked trimer of N-acetyl-D-glucosamine [(NAG)3] When methylamine or 2-hydroxyethylamine was further added, the extent of the cross-link formation was decreased and the derivative where the α-carboxyl group of Leu129 was modified with the amine was newly obtained. On the other hand, when ammonia was added, the β-carboxyl group of Asp119 instead of the α-carboxyl group was mainly amidated. From these results, the presence of a salt bridge between Asp119 and Arg125 besides that between Lys13 and Leu129 is proposed. Enzymatic activities of the derivatives prepared here indicated that the modification of the α-carboxyl group reduced the activity to ̃ 90% of that of native lysozyme. Des-Leu129 lysozyme, which lacks Leu129 also showed ̃ 90% of the activity of native lysozyme. Therefore, the salt bridge between Lys13 and Leu129 may play some role in maintaining the active conformation of lysozyine.

AB - In the cross-linking reaction of lysozyme between Leu129 (α-COO-) and Lys13 (ε-NH3 + using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a side reaction of the peptide bond inversion from α to β between A and Gly102 was greatly reduced by addition of β-(1,4)-linked trimer of N-acetyl-D-glucosamine [(NAG)3] When methylamine or 2-hydroxyethylamine was further added, the extent of the cross-link formation was decreased and the derivative where the α-carboxyl group of Leu129 was modified with the amine was newly obtained. On the other hand, when ammonia was added, the β-carboxyl group of Asp119 instead of the α-carboxyl group was mainly amidated. From these results, the presence of a salt bridge between Asp119 and Arg125 besides that between Lys13 and Leu129 is proposed. Enzymatic activities of the derivatives prepared here indicated that the modification of the α-carboxyl group reduced the activity to ̃ 90% of that of native lysozyme. Des-Leu129 lysozyme, which lacks Leu129 also showed ̃ 90% of the activity of native lysozyme. Therefore, the salt bridge between Lys13 and Leu129 may play some role in maintaining the active conformation of lysozyine.

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