General Acid Catalysis Lysozyme

Nucleophilic participation by the neighbouring acetamido-group in the substrate has also been postulated, but substrates lacking this group have been developed which give enzymatic rates (Raftery and Rand-Meir, 1968). It is therefore not necessary to invoke such participation to explain the rates attained with the enzyme. 

The binding site of lysozyme will accommodate six hexose residues (A-F). The carboxyl groups of glutamic acid-35 and aspartic-52 are located between sites D and E. The lactyl groups at C-3 of the NAM residues cannot fit in sites A, C, and E. Therefore NAM residues must be located at sites B and D. Cleavage occurs at the reducing end of the NAM residue in site D (the bond being broken is between residues D and E). 

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 

At the time the mechanism shown in [60] was proposed it was not known whether it was even chemically reasonable. Bimolecular general acid catalysis involving proton transfer in the transition state [equation (45)] had never been observed in the hydrolysis of glycosides or simple acetals. Bronsted and Wynne-Jones (1929) had 

Prior to 1967 acetal hydrolysis had been found to be a specific-acid catalysed reaction with the accepted mechanism [equation (46)] involving fast pre-equilibrium protonation of the acetal by hydronium ion, followed by unimolecular rate-determining decomposition of the protonated intermediate to an alcohol and a resonance stabilized carbonium ion (Cordes, 1967). An A-1 mechanism was supported by an extremely large body of evidence, but it appeared unlikely that such a mechanism could expledn the 

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On the basis of the above, the rate acceleration afforded by lysozyme appears to be due to (a) general acid catalysis by Glu (b) distortion of the sugar ring at the D site, which may stabilize the carbonium ion and the transition state) and (c) electrostatic stabilization of the carbonium ion by nearby Asp. The overall for lysozyme is about 0.5/sec, which is quite slow (Table... 

GENERAL ACID CATALYSIS AND ELECTROSTATIC STABILIZATION IN THE CATALYTIC REACTION OF LYSOZYME... 

The commonly accepted mechanism for the catalytic reaction of lysozyme is the so-called general acid catalysis mechanism. 

In order to really assess the magnitude of the electrostatic effect in lysozyme on a microscopic level it is important to simulate the actual assumed chemical process. This can be done by describing the general acid catalysis reaction in terms of the following resonance structures ... 

Highly-efficient general acid catalysis of acetal hydrolysis is involved in the reactions of glycosidase enzymes such as lysozyme (Dunn and Bruice, 1973)... 

General acid catalysis by glutamic acid-35 represents at present the mechanism for lysozyme best able to explain the kinetic and structural data. For it to occur, however, distortion of the hexose ring in subsite D to a half-chair must take place so that relief of strain in the transition state will make bond breaking sufficiently easy. A question that must be answered for this picture to be tenable is whether relief of strain can so greatly facilitate bond breaking when the carbonium ion is a glycosyl ion [see also the discussion in Fife (1972) and Atkinson and Bruice (1974)]. 

Lysozyme makes use of covalent catalysis and general acid catalysis as it promotes two successive nucleophilic displacement reactions. 

Catalysis by lysozyme is caused by three main factors (Figure 1) (1) general acid catalysis by glutamic acid residue 35, located in the proximity of the glycosidic bond, initiates the formation of a carbonium... 

Bell-shaped pH-rate profiles are obtained in lysozyme reactions (Rupley et al., 1967) which are consistent with direct involvement of two groups in the reaction. However, bell-shaped pH-rate constant profiles are also observed in the hydrolysis of benzaldehyde disalicyl acetals, and in the case of p-nitrobenzaldehyde o-carboxyphenyl p-carboxyphenyl acetal only one carboxyl group can participate. One should then take care in postulating bifunctional catalysis in the lysozyme reaction, since the observed kinetics and the rate enhancements are explicable in terms of a chemically simpler mechanism (general acid catalysis by glutamic acid-35 along with release of ground state... 

Warshel and collaborators (Warshel and Sussman, 1986 Warshel et al., 1988) developed the empirical valence bond method for obtaining free-energy differences and activation free energies. The effects of Gly-to-Ala mutations in trypsin were accurately simulated. This method was earlier applied to calculation of the potential surface for general acid catalysis of a disaccharide in solution and bound to lysozyme (Warshel and Weiss, 1980). 

The elucidation of the X-ray structure of lysozyme provided the first direct information about the structure of an enzyme-substrate complex (Blake et al., 1967). This and other biochemical studies led to the proposal of three major catalytic factors general acid catalysis, steric strain and electrostatic stabilisation. It was found that the effect of steric strain is unlikely to be important because it can be relaxed by small shifts in the substrate and enzyme geometries (Warshel and Levitt, 1976 Pincus et al., 1977). This point was further established by quantitative FEP calculations (Warshel, 1991). On the other hand, it was found that electrostatic interactions play a very important role in the enzymatic reaction. The total electrostatic stabilisation of the carbonium transition state, relative to the corresponding stabilisation in solution, was found to be 29 kJ/mol (cf. Figure 12., Warshel, 1978). This value appears to account... 

Most of the quantitative work in this area has been done with putative models for lysozyme action. Thus, synchronous acetamido group participation and intramolecular general acid catalysis was held to be responsible for the modest rate enhancement of compound LXI compared with the p-nitrophenyl compound or salicyl glucoside... 

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