Assignment of catalytically essential cysteine residues in aspartase by selective chemical modification with N-(7-dimethylamino-4-methylcoumarynyl)maleimide.
N-(7-Dimethylamino-4-methylcoumarynyl)maleimide (DACM), a fluorescent reagent for sulfhydryl groups, was employed to determine the functionally essential cysteine residues in aspartase from Escherichia coli. Analysis of the tryptic peptides containing DACM-labeled residues by reverse phase HPLC revealed that Cys-140 and Cys-430 were selectively modified, among 11 residues whose loci were recently determined by a DNA sequencing study (Takagi, J.S., et al. (1985) Nucl. Acids Res. 13, 2063-2074). When the modification was carried out in the presence of Mg2+ and L-aspartate, the enzyme activity remained unchanged and no cysteine residue was modified. This suggests that two cysteine residues are located at the L-aspartate binding site and that at least one of them is involved in the catalytic reaction.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000642:Ammonia-Lyases / Q000378:metabolism*; D001220:Aspartate Ammonia-Lyase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014357:Trypsin
D000596:Amino Acids; D008301:Maleimides; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D014357:Trypsin; D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase; D003545:Cysteine