|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1993 : ¥1,700,000 (Direct Cost : ¥1,700,000)
The mechanism for the high efficiency of transglycosylation of lysozyme seems not to have been fully explained. We attempted to clarify the reaction mechanism of lysozyme-catalyzed reaction as follows ; 1.Enzyme Activity of Avian Egg-White Lysozyme : In turkey, peafowl, and quail lysozymes, Asp101-Met105 in the loop region of hen lysozyme (HEL) were replaced by other amino acids. The reaction time-courses of each lysozyme with (GlcNAc) _5 were exhibited different profiles from those of HEL,lowering the substrate affinity at subsite A and/or B.The time-courses of guinea-hen lysozyme (GHL), which replaced Arg114 in subsite F of HEL by His, was the same profile as those of HEL,suggested that His114 in GHL participated in binding of sugar residue at subsite F in the same manner as Arg114 in HEL.2.Enzyme Activity of His114-modified GHL : His114 in GHL was modified by DEP and monoiodoacetic acid. The projiles of time-courses of modified GHL deffered greatly from those of native lysozyme. The time-courses of modified GHL indicated that modified GHL may have increased rate of hydrolysis in comparison with that of native lysozyme. 3.Binding Mode of (GlcNAc) _6 for Trp108 Ester GHL : The resonance of C2H of His114 in ^1H-NMR spectrum is suitable as a probe for investigation of substrate binding at subsite E and F.The modified lysozyme in which an ester linkage had been formed between Glu35 and Trp108 (Trp108 ester GHL) showed no significant activity, and unaffected the environment around subsites. The interaction of (GlcNAc) _6 with modified lysozyme was studied by ^1H-NMR spectra. The resonance of C2H of His114 in Trp108 ester GHL did not change upon the addition of (GlcNAc) _6. The results indicate that the substrate binds to left side of subsite E and F.On the basis of those results, the high efficiency of transglycosylation of lysozyme can be presumed by the formation of a 2 : 1 substrate-acceptor-enzyme complex.