Molecular Dynamics Simulation of Ester‐Linked Hen Egg White Lysozyme Reveals the Effect of Missing Backbone Hydrogen Bond Donors on the Protein Structure

The three‐dimensional structure of a protein is stabilized by a number of different atomic interactions. One of these is hydrogen bonding. Its influence on the spatial structure of the hen egg white lysozyme is investigated by replacing peptide bonds (except those of the two proline residues) by ester bonds. Molecular dynamics simulations of native and ester‐linked lysozyme are compared with the native crystal structure and with NOE distance bounds derived from solution NMR experiments. The ester‐linked protein shows a slight compaction while losing its native structure. However, it does not unfold completely. The structure remains compact due to its hydrophobic core and a changed network of hydrogen bonds involving side chains.     

Lysozymes: a chapter of molecular biology

Hen egg-white lysozyme was the first enzyme whose tertiary structure could be elucidated. The peptide chain of this enzyme is arranged in two sections, of approximately equal size, that are separated by a deep cleft. Substrates (and inhibitors) are bound in this cleft via hydrogen bonds and are hydrolyzed under the action of Glu 35 and Asp 52, which form the active site of the enzyme. Although the lysozymes, which occur in many species of animals and plants, exhibit differences in their chemical behavior, they have the same qualitative biological activity; quantitatively important differences have been noted which also concern the specificity. Infection of E. coli with bacteriophages gives rise to a lysozyme whose formation is controlled by the phage DNA. The fact that mutated lysozymes are produced when the phages are treated with mutagens opens new fields of research in molecular biology.     

Study of Lysozyme Glycation Reaction by Mass Spectrometry and NMR Spectroscopy

The Advanced Glycation End Products (AGEs) are the causative substances of lifestyle‐habit illness. To elucidate the glycation mechanism of the protein, the reaction of lysozyme with D ‐glucose was analyzed by the fluorescence, TOF‐MS, and ¹³C‐NMR spectroscopy under the physiological condition. The fluorescence intensity of lysozyme in the glycation solution increased proportionally with a reaction time of ten weeks. The MALDI‐TOF‐MS spectra of the reaction solution after two weeks showed a peak at m/z 15066, which indicated the presence of a larger molecule than the native lysozyme (m/z 14331), and new peaks at m/z 30105 (dimer) and 45000 (trimer) were also observed. The spectral analysis supported the assumption of a continuous glycation reaction of D ‐glucose with lysozyme and a 30% transformation of lysozyme to the dimeric form during ten weeks. The ¹³C‐NMR spectra of lysozyme showed six [¹³C]‐labeled signals by the glycation reaction with [¹³C]‐glucose after two weeks of reaction. The combined analysis of TOF‐MS and ¹³C‐NMR spectra uncovered that first products of the glycation reaction of lysozyme with D ‐glucose can be observed already three hours after starting the reaction and that nine D ‐glucose units are attached during ten weeks at 37°.