Preparation and Characterization of an Active Lysozyme Derivative: Kyn 62-Lysozyme1

Abstract

A novel method for the preparation of Kyn 62-lysozyme, in which tryptophan 62 is replaced by kynurenine, is reported. Hen egg-white lysozyme was ozonized in aqueous solution to yield one N'-formylkynurenine residue and deformylated with hydrochloric acid in frozen solution at -10 degrees C. Crude Kyn 62-lysozyme was purified by affinity and Bio Rex 70 chromatography successively. Kyn 62-lysozyme retains affinity for chitin and is essentially an active enzyme with a slightly weakened but distinct catalytic activity. After this modification, the enzyme activity was changed differently depending on the kind of substrate. At the individual optimum pH's, lytic activity was largely retained (80% active), but the catalytic efficiency for hydrolyzing glycol chitin was relatively low (30% active). Lysis of M. lysodeikticus cell suspensions was optimally catalyzed by Kyn 62-lysozyme at pH 6.2 and at 0.088 ionic strength. These values are lower by 1.3 pH unit and 0.04 ionic strength, respectively, than those of intact lysozyme. The optimum pH and ionic strength for the hydrolysis of neutral substrates were scarcely affected. These results suggest the significance of electrostatic interaction in the lysis of lysozyme. Relatively limited loss of activity induced by modification of the 62nd residue, which is thought to participate directly in the binding of the substrate at subsite C, is discussed on the basis of the similarity of side chain structure in tryptophan and kynurenine.

Characterization of five new low‐molecular‐mass trypsin inhibitors from white mustard (Sinapis alba L.) seed

“…Although the Trp residue can be oxidized by chemical and physical agents (e.g. ozone, metal and ionizing radiation) [21,22], the Trp to Kyn44 conversion may be catalysed by endogenous enzyme(s). Note that tryptophan pyrrolooxygenase, catalysing Trp oxidation, was identified in plants, bacteria and mammals [23–26].…”

The Steady State and Time-resolved Fluorescence Studies on the Lysozyme-Ligand Interaction

“…HEWL was purchased from Seikagaku Kogyo Co. Kyn62-lysozyme was prepared with the methods of Yamasaki et al 5 ) Tri-N-acetyl-D-glucosamine «GlcNAch) and L-tryptophan were purchased from Sigma Chemical Co.…”

Fluorescence Spectroscopic Properties of an Active Lysozyme Derivative: Kyn 62-Lysozyme

“…S ) This derivative has fairly high enzymatic activity and an absorption maximum at 360 nm. 4 ) In a previous work, we demonstrated that the binding of (GlcNAc)3 to Kyn 62-lysozyme induced the red shift of tryptophan and kynurenine, producing a UVdifference spectrum with maxima at 293 and 390nm. S ) These findings directed us to shed further light on the active site of lysozyme using spectroscopic characteristics of Kyn 62-lysozyme.…”