title: Lysozyme revisited: crystallographic evidence for distortion of an N-acetylmuramic acid residue bound in site D.
abstract: A structure of the trisaccharide 2-acetamido-2-deoxy-D-muramic acid-beta (1----4)-2-acetamido-2-deoxy-D-glucose-beta (1----4)-2-acetamido-2-deoxy-D-muramic acid (NAM-NAG-NAM), bound to subsites B, C and D in the active-site cleft of hen egg-white lysozyme has been determined and refined at 1.5 A resolution. The resulting atomic co-ordinates indicate that the NAM residue in site D is distorted from the full 4C1 chair conformation to one in which the ring atoms C-1, C-2, O-5 and C-5 are approximately coplanar, and the hydroxymethyl group is positioned axially (a conformation best described as a sofa). This finding supports the original proposals that suggested the ground-state conformation of the sugar bound in site D is strained to one that more closely resembles the geometry required for the oxocarbonium-ion transition state, the next step along the reaction pathway. Additionally, detailed analysis at 1.5 A resolution of the environments of the catalytic residues Glu35 and Asp52 provides new information on the properties that may allow lysozyme to promote the stabilization of an unusually long-lived oxocarbonium-ion transition state. Intermolecular interactions between the N-acetylmuramic acid residue in site D and the lysozyme molecule that contribute to the saccharide ring distortion include: close packing of the O-3' lactyl group with a hydrogen-bonded "platform" of enzyme residues (Asp52, Asn46, Asn59, Ser50 and Asp48), a close contact between the hydroxymethyl group of ring D and the 2'-acetamido group of ring C and a strong hydrogen-bonded interaction between the NH group of Val109 and O-6 of ring D that stabilizes the observed quasi-axial orientation of the -CH2OH group. Additionally, the structure of this complex shows a strong hydrogen bond between the carboxyl group of Glu35 and the beta-anomeric hydroxyl group of the NAM residue in site D. The hydrogen-bonded environment of Asp52 in the native enzyme and in the complex coupled with the very unfavorable direction of approach of the potential carboxylate nucleophile makes it most unlikely that there is a covalent glycosylenzyme intermediate on the hydrolysis pathway of hen egg-white lysozyme.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: N C
authors.initials: NC
authors.lastname: Strynadka
authors.forename: M N
authors.initials: MN
authors.lastname: James
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002645:Chickens; D006860:Hydrogen Bonding; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009112:Muramic Acids / Q000737:chemistry* / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D014961:X-Ray Diffraction
chemical_list: D009112:Muramic Acids; C031651:N-acetylmuramic acid; D009113:Muramidase