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Site‐directed mutagenesis and chemical modification of the two cysteine residues of the UDP‐ N ‐acetylmuramoyl: l ‐alanine ligase of Escherichia coli
Author(s) -
Nosal Florence,
Masson Anne,
Legrand Raymond,
Blanot Didier,
Schoot Bernard,
van Heijenoort Jean,
Parquet Claudine
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00364-0
Subject(s) - alanine , mutagenesis , chemistry , cysteine , site directed mutagenesis , escherichia coli , biochemistry , dna ligase , enzyme , iodoacetamide , substrate (aquarium) , stereochemistry , amino acid , mutation , biology , mutant , gene , ecology
Site‐directed mutagenesis and chemical modification of the two cysteine residues of the MurC l ‐alanine‐adding enzyme from Escherichia coli were undertaken to study their possible role in activity and stability. Their replacement by alanine was not critical for activity. However, C230 played a role in enzyme stability and substrate binding. N ‐Ethylmaleimide alkylation led to monoalkylated and dialkylated proteins. The monoalkylated protein had mostly unmodified C230 residues. The extent of alkylation of C230 paralleled the loss of activity, whereas that of C426 did not. Protection against inactivation by β,γ‐imidoadenosine 5′‐triphosphate implied the involvement of C230 in the ATP binding site.