title: Investigation of the substrate specificity of glutamyl endopeptidase using purified bovine β-casein and synthetic peptides.
abstract: Purified bovine β-casein was digested with glutamyl endopeptidase (GE) at 37 and 50 °C for 4 h. The peptides generated were determined using nano-LC-ESI-qTOF-MS/MS. GE was highly specific and hydrolyzed peptide bonds in β-casein predominantly on the carboxy terminal of Glu and Asp. Pro residues were not preferred, while Met was poorly preferred at the P1' position. Glu-Met hydrolysis was less preferred in comparison to Asp-Met hydrolysis. Five synthetic peptides corresponding to specific sequences in β-casein were incubated with GE at 37 °C to further characterize the substrate specificity. MS analysis of the digestion products indicated that GE hydrolyzed Glu-Ser in Glu-Glu-Ser. Furthermore, hydrolysis of Glu-Met and Glu-Pro was observed. The presence of multiple-phosphorylated Ser residues upstream from the scissile bond did not appear to affect hydrolysis of Glu-Ser. The results herein are relevant to our understanding of the substrate specificity of GE and the peptides that may be expected during the hydrolysis of β-casein.
authors.affiliation: Department of Life Sciences, University of Limerick, Limerick, Ireland.
authors.forename: Phanindra
authors.initials: P
authors.lastname: Kalyankar
authors.forename: Yishen
authors.initials: Y
authors.lastname: Zhu
authors.forename: Gerard
authors.initials: G
authors.lastname: O'Cuinn
authors.forename: Richard J
authors.initials: RJ
authors.lastname: FitzGerald
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D002853:Chromatography, Liquid; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D002364:Caseins; D010455:Peptides; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase