State of Trp 62 in Hen Egg-white Lysozyme Bound with N-Acetylchitooligosaccharides<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

Abstract

In order to clarify the state of Trp 62 in hen egg-white lysozyme [EC 3.2.1.17] bound with N-acetylchitooligosaccharides in relation to the ionization states of side chains present in the substrate binding site, the binding of alpha- and beta-N-acetylglucosamine GlcNAc), alpha- and beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 was studied by use of the change in the tryptophyl circular dichroic (CD) band at 295 nm at various pH values. The effects of these saccharides on the charge-transfer binding of N-1-methylnicotinamide to Trp 62 were also examined. The binding constants, estimated by use of N-1-methylnicotinamide by assuming noncompetitive binding between the saccharides and N-1-methylnicotinamide, were in good agreement with those determined from the CD change at 295 nm. The state of Trp 62 in saccharide-bound lysozyme was found to depend on the chemical structures of the saccharides and their binding orientations. The relation between the state of Trp 62 and the ionization of the catalytic Glu 35, which is far distant from Trp 62, was also confirmed for lysozyme complexes with various saccharides as well as for free lysozyme.