Site‐directed mutagenesis and chemical modification of the two cysteine residues of the UDP‐N‐acetylmuramoyl:l‐alanine ligase of Escherichia coli

FEBS Letters

Parquet, Claudine¹  Masson, Anne¹  Schoot, Bernard²  van Heijenoort, Jean¹  Blanot, Didier¹  Legrand, Raymond²  Nosal, Florence² 

[1] Biochimie Structurale et Cellulaire, CNRS, Bâtiment 430, Université Paris-Sud, 91405 Orsay, France;Biophysics, Hoechst-Marion-Roussel, 93230 Romainville, France

关键词: MurC synthetase;    Cysteine residue;    Site-directed mutagenesis;    N-Ethylmaleimide;    ADPNP;    β;    γ-imidoadenosine 5′-triphosphate;    BSA;    bovine serum albumin;    DTT;    dithiothreitol;    EDTA;    ethylene diaminetetraacetic acid;    ESI;    electrospray ionization;    MS;    mass spectrometry;    LC-MS;    liquid chromatography-mass spectrometry;    MurC;    UDP-MurNAc:l-alanine ligase;    l-alanine-adding enzyme;    MurNAc;    N-acetylmuramic acid;    NEM;    N-ethylmaleimide;    TFA;    trifluoroacetic acid;    SDS;    sodium dodecylsulfate;    WT;    wild-type;   

DOI  :  10.1016/S0014-5793(98)00364-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown