Lysozyme substrate distortion

The more recent structural evidence supports the role of substrate distortion. A trisaccharide in the BCD sites of crystalline hen egg white lysozyme is distorted to the half-chair,229 as is a substrate bound to the Asp-52 — Ser mutant studied in solution by NMR.230 Protein engineering studies on the residues involved support the conclusions.231 A chitobiose bound to a crystalline chitobiase has the equivalent sugar distorted to a sofa.232... 

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 ). ... 

The mechanism of action of lysozyme has been suggested to involve strain in the substrate as a result of binding to the protein.31 The protein requires that the substrate binds to the protein in a distorted state that is close to the transition state of the reaction. The energy for this distortion can arise from the substrate binding energy. This example differs from the entatic state hypothesis because this strain arises from substrate binding and is not a feature of the protein structure. However, the entatic state... 

Note that the entatic state differs from the situation where the substrate itself is strained by virtue of binding to the protein. Such a case is also found in lysozyme, where the energy of holding the substrate (a saccharide) is used partly to distort the substrate toward its transition state, thus lowering the activation energy. 

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. 

Fig. 6. Proposed reaction mechanisms by which lysozyme catalyzes the cleavage of a polysaccharide substrate. Scheme I is that proposed by Post and Karplus (1986) and indicates the possibility of cleavage with no prior assumption of distortion of the sugar ring at site D. Scheme II is that originally developed by Blake, Johnson, Phillips, and co-workers and involves ring distortion as a critical step. (Reproduced with permission from Post and Karplus, 1986.)...

Kuroki, R., Weaver, L. H., and Matthews, B. W. A covalent enzyme-substrate intermediate with saccharide distortion in a mutant T4 lysozyme. Science 262, 2030-2033 (1993). 

Fig. 4.5. Structure of an hexasaccharide that can bind at the active center of lysozyme. Upon binding to the enzyme, the sugar ring D of the substrate becomes distorted and catalysis proceeds through the promotion of an oxocarbonium ion (see p. 228). This results in a polar transition state. However, an important feature of enzyme is the capacity to stabilize (neutralize) the enzyme-substrate complex by electrostatic interactions with amino acid residues at the active site.

Figure 2.1 The polysaccharide molecule found in the walls of certain bacterial cells is the substrate broken by the lysozyme molecule. The polysaccharide consists of alternating residues of two kinds of amino sugar N-acetylglucosamine and N-acetylmuramic acid. In the portion of polysaccharide chain shown here A, C andE are N-acetylglucosamine residues B, D and Fare N-acetylmuramic acid residues. Ring D is distorted when adsorbed on the enzyme. The position attacked is indicated by the arrows...

---