STRUCTURAL-CHANGES OF ACTIVE-SITE CLEFT AND DIFFERENT SACCHARIDE BINDING MODES IN HUMAN LYSOZYME CO-CRYSTALLIZED WITH HEXA-N-ACETYL-CHITOHEXAOSE AT PH 4.0

Human lysozyme was co-crystallized with hexa-N-acetyl-chitohexaose, (G lcNAc)(6), at pH 4.0 and 4.0 degrees C in a new orthorhombic form, whe re two protein molecules, MOL1 and MOL2, were contained in an asymmetr ic unit. The three-dimensional structure was refined to an R-factor of 17.0% at 1.6 Angstrom resolution. It was found that (GlcNAc)(6) had a lready been cleaved to (GlcNAc)(4) and (GlcNAc)(2). In MOL1, (GlcNAc)( 4) was bound to the A, B, C and D subsites, and the binding sites of ( GlcNAc)(2) were close to the E and F subsites proposed on the basis of model building by Phillips and his colleagues. In MOL2, only the (Glc NAc)(4) moiety could be found in the A, B, C and D subsites. Significa nt shifts of the backbone atoms were observed in the region of residue s 102 to 120, Which composed one side of the wall of the active site c left. Consequently, the active cleft, with respect to the saccharide b inding sites A, B and C, is narrower in both protein molecules. The re sidues 109 to 111 in site D of MOL1 are moved toward saccharide residu e D, whereas those of MOL2 are only slightly shifted. In spite of thes e facts, the saccharide residues in site MOL1 and MOL2 are moved insid e of the cleft. The distribution of water molecules and the hydrogen b ond network in site D differ between the structures of MOL1 and MOL2. These structural changes in the active site cleft may be responsible f or accommodating the substrate and releasing the products of hydrolysi s. These results suggest that the three-dimensional structures of MOL1 and MOL2 remain in intermediate states between a transition state and an enzyme/product complex state.