SITE-DIRECTED MUTAGENESIS AND CHEMICAL MODIFICATION OF THE 2 CYSTEINERESIDUES OF THE UDP-N-ACETYLMURAMOYL-L-ALANINE LIGASE OF ESCHERICHIA-COLI

Site-directed mutagenesis and chemical modification of the two cystein e residues of the MurC L-alanine-adding enzyme from Escherichia coli w ere 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-Ethylm aleimide 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 o f C426 did not. Protection against inactivation by beta,gamma-imidoade nosine 5'-triphosphate implied the involvement of C230 in the ATP bind ing site. (C) 1998 Federation of European Biochemical Societies.