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 1H-15N 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 1H-15N HSQC NMR spectroscopy identified the residues which were most affected upon interaction with [MoO4]2 −. 95Mo 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 1H NMR, further supporting a hydrolytic mechanism where polarisation of the carbonyl is followed by internal nucleophilic attack on the Asp residue.

Hen egg white lysozyme (HEWL) was selectively hydrolyzed in the presence of oxomolybdate(VI) at the Asp18-Asn19, Asp48-Gly49, Asp52-Trp53 and Asp101-Gly102 peptide bonds. Molecular interactions between [MoO4]2 − and HEWL were studied by circular dichroism, 1H-15N Heteronuclear Single Quantum Correlation and95Mo NMR spectroscopy which identified the monomeric [MoO4]2 − as catalytically active species.Figure optionsDownload as PowerPoint slide