Lysozyme configurations

FIGURE 6.3. The strain hypothesis for the catalytic reaction of lysozyme. The enzyme is assumed to destabilize the chair geometry by pushing the ground state substrate toward the sofa configuration. This ground-state destabilization effect is supposed to reduce Ag M. 

In order to make an effective use of the VB formulation we have to calibrate the relevant parameters using reliable experimental information. The most important task is to obtain the relevant a-. Since the a s represent the energy of forming the different configurations in the gas phase at infinite separation between the given fragments, it is natural to try to obtain them from gas-phase experiments. In the case of the catalytic reaction of lysozyme one can compile the relevant information from the available gas-phase experiments (Table 6.1) and use it to determine the a s. 

FIGURE 6.10. Comparing the energetics of the EVB configurations in solution and in the active site of lysozyme. The calculations were done by using the PDLD and related models (Refs. 6 and 7) and they represent a study of a stepwise mechanism. The energetics of a more concerted pathway (e.g., that of Fig. 6.9) is almost identical to that of the stepwise mechanism and correlated in a similar way with the electrostatic effect of the protein. 

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. 

A pathway (Scheme I) (8.9) for the hydrolysis of oligoglycosides by lysozyme that differs from the previously accepted mechanism (Scheme II) (3,10-12) is described in this section. The alternative pathway, suggested by results of a 55-ps MD simulation of the lysozyme (GlcNAc)e complex (1), is consistent with the available experimental data and with stereoelectronic considerations. Experimental data have demonstrated that Glu 35 and Asp 52 are essential, as shown by recent site-directed mutagenesis results (13.) which corroborate chemical modification studies (3.14 and references cited therein), and that the reaction proceeds with retention of configuration at Ci Q and references cited therein). A fundamental feature of the alternative pathway is that an endocyclic bond is broken in the initial step, in contrast to the exocyclic bond cleavage in the accepted mechanism. 

Cellulose, the most abundant of all biopolymers, is extremely stable but is attacked by a host of bacterial and fungal (3-glycanases.96 Animals do not ordinarily produce cellulases but some termites do.97 Cellulase structures are varied, being represented by 10 of 57 different glycosylhydrolase families.98 Most, like lysozyme, retain the P configuration in their products but some invert.98 100... 

Lysozyme and /3-galactosidase, which are both glycosidases, catalyze very similar reactions. Both enzymes are found to catalyze the alcoholysis of their polysaccharide substrates with retention of configuration at the C-l carbon (equation 8.25).14-17 This is consistent with the evidence presented in Chapter 7, section C3, that there is at least one (but probably only one) intermediate on the reaction pathway. However, kinetic isotope data are consistent with the interpretation that the intermediate in the reaction of /3-galactosidase is covalent and that there are two successive SN2 displacements, whereas the intermediate with lysozyme is a bound carbonium ion formed in an SN1 reaction (Chapter 16). The carbonium ion, unlike an analogous one in solution, reacts stereospecifically on the enzyme. Thus, the stereochemical evidence by itself has given no indication of the nature of the intermediate. 

Hen egg white lysozyme is a small protein of Mr 14 500 and 129 amino acid residues. This enzyme was introduced in Chapter 1, where it was pointed out that examination of the crystal structure of the enzyme stimulated most of the solution studies. Hen egg white lysozyme has the distinction of being the first enzyme to have had its structure solved by x-ray crystallography.207 It is an atypical member of the hexosaminidase class of glycosyl transfer enzymes. It catalyzes the hydrolysis of substrates with retention of stereochemistry. T4 lysozyme was for many years thought to have the same fold and mechanism of lysozyme, despite there being no sequence homology. But it has now been found that the T4 enzyme has inversion of configuration and so operates by a different mechanism.208,209 A mechanism proposed for the enzymatic reaction was based on the structure of the... 

TABLE 6.1. Gas-Phase Enthalpies that Can Be Used to Determine the Energies of the Different Configurations Involved in the Catalytic Reaction of Lysozyme ... 

Lysozyme hydrolyses a polysaccharide present in bacterial cell walls that is a / -(l—>4) linked polymer of alternating IV-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues. The enzyme only attacks the NAM—>NAG linkage. Certain lysozymes also attack chitin, the / -(l- 4) linked linear polymer of NAG (9). It was demonstrated by Rupley (5), using oligosaccharides of NAG, that the Ci-O linkage was specifically cleaved and / -configuration was retained. 

Studies of the stereochemical course of the transglycylation reactions catalysed by lysozyme show retention of configuration at Cl in the product. This can be attributed most economically to the intervention of an acylal intermediate in the mechanism (see Scheme 11.21). A similar result is found in the alkaline phosphatase-catalysed solvolysis of isotopomeric phospho-esters which indicates a double inversion via a phospho-enzyme intermediate (Scheme 11.22). 

It is worthwhile to present some examples of the types of results available from Monte Carlo simulations of peptide solvent systems. In Figure 6 we present the convergence of the energy of hydration of the lysozyme crystal over a million configurations. 

Figure 6. The average energy of the water of hydration of the triclinic lysozyme crystal as a function of the number of configurations generated in the Monte Carlo simulation. The upper curve (A) corresponds to the cumulative statistical average, and the lower curve fU gives the statistical average over sequential sets of 5,000...

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