TY - JOUR
T1 - Investigation of the substrate specificity of glutamyl endopeptidase using purified bovine β-casein and synthetic peptides
AU - Kalyankar, Phanindra
AU - Zhu, Yishen
AU - O'Cuinn, Gerard
AU - Fitzgerald, Richard J.
PY - 2013/4/3
Y1 - 2013/4/3
N2 - 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.
AB - 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.
KW - bovine β-casein
KW - glutamyl endopeptidase
KW - LC-MS/MS
KW - substrate specificity
KW - synthetic peptides
UR - http://www.scopus.com/inward/record.url?scp=84875778622&partnerID=8YFLogxK
U2 - 10.1021/jf305274e
DO - 10.1021/jf305274e
M3 - Article
C2 - 23473379
AN - SCOPUS:84875778622
SN - 0021-8561
VL - 61
SP - 3193
EP - 3204
JO - Journal of Agricultural and Food Chemistry
JF - Journal of Agricultural and Food Chemistry
IS - 13
ER -