title: Substrate preferences of glutamic-acid-specific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching.
abstract: The substrate preferences of the easily available Glu/Asp-specific enzymes from Staphyllococcus aureus (V8), Bacillus licheniformis and Streptomyces griseus have been extensively investigated using a series of synthetic peptide substrates, containing an N-terminal anthraniloyl group and a 3-nitrotyrosine close to the C-terminus, allowing the fluorimetric monitoring of substrate hydrolysis by the decrease in intramolecular quenching. All three enzymes hydrolysed Glu-Xaa peptide bonds approximately 1000-fold faster than Asp-Xaa bonds and they are consequently more appropriately termed Glu-specific enzymes. The difference in kcat/Km for the hydrolysis of substrates with Glu and Asp is primarily due to a difference in kcat. The enzymes appear to hydrolyse all types of Glu-Xaa bonds, although those with Xaa as Asp and, in particular, Xaa as Pro, are hydrolysed with very low rates. The influence of the nature of the amino acid residues at the substrate positions P2, P3, P4, P'1 and P'2 has been determined and it is shown that the enzyme from S. griseus exhibits the most narrow substrate preference. The results are useful in connection with fragmentation of proteins for sequencing purposes as well as for cleavage of fusion proteins.
authors.affiliation: Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark.
authors.forename: K
authors.initials: K
authors.lastname: Breddam
authors.forename: M
authors.initials: M
authors.lastname: Meldal
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D012697:Serine Endopeptidases; C069136:glutamyl endopeptidase II