Lysozyme reaction

FIGURE 16.36 The Q-O bond, not the O-C4 bond, is cleaved in the lysozyme reaction. from H9 0 is thus incorporated at the position. 

The mechanism of the lysozyme reaction is shown in Figures 16.36 and 16.37. Studies using O-enriched water showed that the Ci—O bond is cleaved on the substrate between the D and E sites. Hydrolysis under these conditions incorporates into the Ci position of the sugar at the D site, not into the oxygen at C4 at the E site (Figure 16.36). Model building studies place the cleaved bond approximately between protein residues Glu and Asp. Glu is in a nonpolar or hydrophobic region of the protein, whereas Asp is located in a much more polar environment. Glu is protonated, but Asp is ionized... 

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 reduction of the rate of hydrolysis due to lowering the temperature 45 C in the solution containing 7 M NH4NO3 is Ah2o/ nh4N03,7m = 4.78, with an activation energy of 5 kcal mol . This solvent is not suitable for low-temperature studies of the lysozyme reaction. 

Transglycosidation with retention of configuration (Chipman and Sharon, 1969) would be more difficult to explain if an open-chain carbonium ion were formed in lysozyme reactions, necessitating an equilibrium reaction with free aldehyde. It seems unlikely, however, that a cyclic carbonium ion intermediate could have a sufficiently long lifetime to react with a saccharide molecule that can bind to the enzyme only after displacement of the leaving group in a fairly aqueous environment (above discussion). Therefore, the concept of a cyclic carbonium ion also presents difficulties for interpretation and should not be accepted uncritically. 

If aspartic acid-52 acts as a nucleophile in lysozyme reactions a glycosyl enzyme intermediate will be formed [60]. There is no evidence, kinetic or otherwise, for substituted enzyme intermediates, but rapid breakdown might preclude attainment of detectable concentrations. Formation of a substituted enzyme could explain the observed retention of configuration at the anomeric carbon in transglycosidation reactions, provided backside attack in a subsequent reaction is chemically reasonable. It has therefore been important to attempt to understand the chemistry of acylal hydrolysis so as to assess the properties that would be expected of an acylal intermediate in reactions catalysed by the enzyme. 

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... 

Kirby, A. J. Mechanism and stereoelectronic effects in the lysozyme reaction. CRC Crit. Rev. Biochem. 22, 283-315 (1987). 

Kirby, A. J. (1987) Mechanism and Stereoelectronic Effects in the Lysozyme Reaction, Crit Rev. Biochemistry 11, 283-315. 

It has been shown that the structure of the enzyme-substrate complex undergoes rearrangement in the rate-determining step of the lysozyme reaction (29). This rearrangement may require the mobility associated with completion of the water monolayer. It is also possible that a network of water molecules participates in the catalytic process. We favor the former alternative. Regardless of the explanation, it is important that not much water is needed for enzymatic activity and that only the strongly interacting sites must be filled before activity is observable. 

Earlier suggestions of stereoelectronic control of acetal cleavage for the lysozyme reaction Gorenstein, D. G., Findlay, J. B., Luxon, B. A., Kar, D. (1977). Stereoelectronic control in carbon-oxygen and phosphorus-oxygen bond breaking processes. Ah initio calculations and speculations on the mechanism of action of ribonuclease A, staphylococcal nuclease, and lysozyme. Journal of the American Chemical Society, 99, 3477. 

Lysozyme from egg white (Seikagaku Co., Ltd. Japan), crystallized 5 times, was mixed homogeneously with D-glucose at a ratio of 2 1 in a small quantity of water. The whole mixture was lyophilized and incubated at 50°C and 75%RH for 7 days. After the reaction, the browned lysozyme reaction mixture was dialyzed against water for 3 days at 4°C to remove the reaction products of low molecular weight, and then lyophilized again to obtain the nondialyzable modified lysozyme. Modified lysozyme hydrolysate was prepared by the reaction of pepsin (3 h) and pancreatin (20 h) at 37°C as described by Kato et al (1986). 

Combined quantum/classical treatment of molecular systems was introduced first in the pioneering paper of Warshel and Levitt. This paper contains the main features of the QM/MM approach widely used (with modifications) in other works on modeling organic systems. The authors considered a relatively large biological enzyme-substrate complex to study the mechanism of the lysozyme reaction of cleavage of hexasacharides with particular emphasis on factors that contribute to the stabilization of the carbonium ion intermediate. 

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