Selective hydrolysis of hen egg white lysozyme at Asp-X peptide bonds promoted by oxomolybdate.
The activity of oxomolybdate(VI) towards hen egg white lysozyme (HEWL) was examined under physiological and slightly acidic pH conditions. Purely hydrolytic cleavage of HEWL in the presence of 10 to 100 mM of oxomolybdate(VI) after incubation at pH 5.0 and 60 °C for 2 to 7 days was observed in SDS-PAGE experiments. Four cleavage sites, which all occurred at Asp-X sequences and included the Asp18-Asn19, Asp48-Gly49, Asp52-Trp53 and Asp101-Gly102 peptide bonds, were identified with Edman degradation. The molecular interaction between [MoO4](2-) and HEWL was studied by circular dichroism (CD) and (1)H-(15)N heteronuclear single quantum correlation (HSQC) NMR spectroscopy. CD spectroscopy revealed a significant decrease in the α-helical content of HEWL upon addition of oxomolybdate, while (1)H-(15)N HSQC NMR spectroscopy identified the residues which were most affected upon interaction with [MoO4](2-). (95)Mo NMR measurements, performed on oxomolybdate solutions containing HEWL, identified the monomeric [MoO4](2-) form as active species in the hydrolytic reaction. The hydrolysis of the Asp-Gly model peptide in the presence of oxomolybdate(VI) was studied by (1)H NMR, further supporting a hydrolytic mechanism where polarisation of the carbonyl is followed by internal nucleophilic attack on the Asp residue.
Department of Chemistry, KU Leuven, Belgium.
Karen
K
Stroobants
Department of Chemistry, KU Leuven, Belgium.
Phuong Hien
PH
Ho
Department of Microbiology and Immunology, KU Leuven, Belgium.
Eva
E
Moelants
Department of Microbiology and Immunology, KU Leuven, Belgium.
Paul
P
Proost
Department of Chemistry, KU Leuven, Belgium. Electronic address: Tatjana.Vogt@chem.kuleuven.be.
Tatjana N
TN
Parac-Vogt
D020816:Amino Acid Motifs; D000818:Animals; D002645:Chickens; D004527:Egg Proteins / Q000737:chemistry*; D006868:Hydrolysis; D008982:Molybdenum / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D059748:Proteolysis
D004527:Egg Proteins; D008982:Molybdenum; D009113:Muramidase
Hen egg white lysozyme; Hydrolysis; Metal complexes; Oxoanions; Oxomolybdate; Proteins