The present review is focused on the main achievements realized in the lysozyme research field since the meeting held in 1972 to commemorate the fiftieth anniversary of the discovery of this enzyme. Despite of extensive structural, physico-chemical, crystallographic, genetic, immunological and evolutionary studies devoted to lysozymes, their biological role is still not exactly known.

What's new in lysozyme research? Always a model system, today as yesterday.

Lysozyme revisited : crystallographic evidence for distortion of an n-acetylmuramic acid residue bound in site d

The energetics of association of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) and its beta(1,4) oligomers have been measured using isothermal titration calorimetry. Association constants of 0.4, 5.3, 11.1, 12.3, and 19.1 mM-1 and enthalpies of binding of -6.1, -15.6, -19.4, -19.3, and -18.2 kcal mol-1 were obtained at 26 degrees C for the titration of WGA with GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, respectively. The term T delta S was always of negative value, indicating that the binding process is enthalpically driven. Titrations of WGA performed at pH 4.5 did not differ significantly from those performed at pH 7.0, suggesting that no groups with a pKa in this range are directly involved in the binding event. Also, performing the titration in a buffer system with a higher enthalpy of protonation did not change the enthalpy of binding confirming that there is no net protonation or deprotonation when WGA binds GlcNAc residues at pH 7. A model of four independent binding sites was found to adequately describe the binding curves, except in the case of (GlcNAc)4 which exhibited positive cooperativity. The energetic values are discussed within the context of the structure of the WGA-(GlcNAc)2 complex.

Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers.

Hen egg white lysozyme was the first enzyme whose structure was determined by X-ray crystallography The proposed mechanism based on this structure involves the distortion of the saccharide residue (2-acetamido-2-deoxy-D-muramic acid, NAM) in the natural substrate (an alternating beta (1 leads to 4) linked oligomer of 2-acetamido-2-deoxy-D-glucose (NAG) and NAM residues) bound to site D in the binding cleft The importance of substrate distortion has prompted numerous enzymatic, chemical, theoretical, and physical studies, but there is little direct crystallographic evidence on the conformation of a NAM residue bound at site D We now present the X-ray structure of the non-hydrolysed trisaccharide NAM-NAG-NAM bound in subsites B, C, D Our interpretation of the 25-A resolution difference map does not involve distortion of this residue in site D Comparison with the structure of the delta-lactone derived from tetra N-acetylchitotetraose (NAG)3NAL) bound to lysozyme suggests we may be looking at a Michaelis complex

X-ray crystallography of the binding of the bacterial cell wall trisaccharide NAM-NAG-NAM to lysozyme.

(1987). Mechanism and Stereoelectronic Effects in the Lysozyme Reactio. Critical Reviews in Biochemistry: Vol. 22, No. 4, pp. 283-315.

Mechanism and stereoelectronic effects in the lysozyme reaction.

The binding of the trisaccharide (2-acetamido-2-deoxy-D-muramic acid)-β(1→4)-(2-aceta-mido-2-deoxy-D-glucosyl)-β(1→4)-(2-acetamido-2-deoxy-D-muramic acid) to subsites B, C, and D in lysozyme has been studied by 1H nuclear magnetic resonance methods. In particular, the coupling constant between H1 and H2 of the reducing saccharide bound in subsite D has been determined. The coupling constant for the bound saccharide indicates that the dihedral angle between C1 and C2 for the reducing saccharide is not significantly changed upon binding to lysozyme. This result is discussed in terms of other evidence for the role of distortion of the saccharide bound in subsite D in the lysozyme-catalyzed hydrolysis of cell wall oligosaccharides.

Gregg Weisz

Papers

The nuclear magnetic resonance determination of the conformation of saccharides bound in subsite D of lysozyme.