Lysozyme substrate conformation

Since use of increasing amounts of cosolvents as antifreeze could perturb the conformation and thus the activity of lysozyme, a number of experiments were carried out to try to determine conditions for investigating lysozyme reactions and lysozyme-substrate intermediates in cooled mixed solvents as a preliminary to similar investigation by X-ray diffraction on crystals. Work began with an estimate of the solubility of... 

The initial step in the alternative hydrolysis mechanism is protonation of the ring 0i by Glu 35 (Scheme I). Cleavage of the endocyclic C1-O5 bond forms the acyclic oxocarbonium ion intermediate, which is stabilized by Asp 52. Attack by water, cleavage of the C1-O4 bond, and ring closure then lead to the observed products. Existing experimental data on lysozyme hydrolysis are consistent with Scheme I (see references in Post and Karplus ( )). Moreover, distortion of the ring in site D is not required and the antiperiplanar orientation of an exocyclic O4 lone pair orbital relative to the cleaved C1-O5 bond found in the simulation (see section on "Enhancement of a Substrate Conformation Optimum for Catalysis") is in accord with stereoelectronic requirements (1 ). ... 

A half-chair conformation of a crystalline monosaccharide has not been observed. A half-chair conformation for the fourth 2-acetamido-2-deoxy-/3-D-glucopyranosyl residue (residue D) in the lysozyme substrate has not been detected, although, on the basis of model fitting, its presence has been suggested (see p. 96). 2-Acetamido-2-deoxy-/3-D-glucosyl groups were added to a molecular model constructed by use of data obtained from the nature of the enzyme-trisaccharide complex it was implicit that the lifetime of the half-chair conformation would be quite short. 

An important question regarding peptides and proteins is concerned with the equilibria among several conformational states. It has been suggested that enzyme function may be linked to the occurrence of particular conformations in solution.24 377 A mechanism recently proposed for the hydrolysis of oligosa-charides by the enzyme lysozyme, for example, is based on the observation of specific substrate and enzyme sidechain conformations in a molecular dynamics simulation of a lysozyme-substrate complex.378 Also, local conformational equilibria and the barriers between conformations are important in determining the rates and mechanisms of folding and rebinding processes. 

In addition to x-ray elucidation of the structure of the crystalline enzyme, the structure of a crystalline complex of lysozyme and tri(N-acetylglucosamine) was determined (Phillips, 1966). The trisaccharide occupied sites A, B, and C. Assuming that binding of a hexamer (adding hexose residues D, E, and F) would not change the conformation of the enzyme, the conformations of the substrate at... 

One result from the analysis of the MD simulation was the proposal of a new enzymic pathway for hydrolysis by lysozyme. We begin with a description of the alternative mechanism, and the basis on which it was proposed. The energetics of the individual GlcNAc units in the lysozyme cleft are then presented, followed by a graphical representation of the correlation between the atomic fluctuations of the substrate and those of the enzyme. Of particular interest is the fact that the binding interactions stabilize a bound state conformation for the two glycosides involved in hydrolysis that is optimum for catalysis by the alternative mechanism and which differs from the conformations of the other glycosides. These conformational features are described in the final two sections. 

In the crystalline state these two forms are not isomorphous, and adding substrate to ConA crystals causes them to shatter.48 (This is a common observation and occurs even in hemoglobin crystals.) The differences between the conformers in the ConA part of the complex are not fully known, but there is considerable rearrangement of the protein. The rates of the reaction are fast, and so in solution the protein must be able to fluctuate readily between its different forms. Comparison with lysozyme (earlier in this article) shows that when lysozyme binds its inhibitors, a conformational change does occur, but it is not so gross that in the solid state shattering of crystals occurs. 

Both glycosidases (61) and amylases (62) are inhibited by certain lactones, as is lysozyme, D-glucono-1,5-lactone, for example, is presumed to inhibit amylase by acting as a transition state analog because it closely approximates a half-chair conformation. However, as stated by Laszlo et al. (62), lactone inhibition cannot establish whether distortion of substrate occurs during binding, as in lysozyme, or after bond splitting to form the carbonium ion, as in proton catalysis. 

It has been pointed out in Chapter 2 (p. 39) (see also 1-4) that there is good evidence which indicates that in the hydrolysis of B-glycosides by lysozyme, the substrate must take a boat conformation in order to produce the half-chair oxocarbonium ion 0+2- -3i)- Lysozyme is therefore a good example which provides evidence that stereoelectronic effects play a key role in enzymic hydrolysis. 

The internal motion of T4 lysozyme in the crystal was interpreted as an inter-domain motion corresponding to opening and closing of the active site cleft (Weaver et al., 1989). Hinge-bending and substrate-induced conformational transition in T4 lysozyme in solution were confirmed in a study by site-directed labelling (Mchaourban et al., 1997). 

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