Lysozyme configurational studies

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. 

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. 

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... 

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). 

Lysozyme has two catalytic groups at the active site Glu 35 and Asp 52 (Figure 24.9). Once it was determined that the enzyme-catalyzed reaction takes place with retention of configuration at the anomeric carbon, it could be concluded that it cannot be a one-step Sn2 reaction the reaction must involve at least two steps and, therefore, must form an intermediate. Although lysozyme was the first enzyme to have its mechanism studied— and the mechanism has been studied extensively for almost 40 years—only recently have data been obtained that support the mechanism shown in Figrue 24.9 ... 

In our new study, we put two randomly oriented OPLS-AA lysozyme molecules and 484 TIP4P-Ew water molecules h = 0.3) in a box. After an energy minimization of 5000 steps with a steep descent algorithm, the system is equilibrated in a NPT ensemble [51]. Many simulations are then performed at different T (in the interval 180-280K, with steps of lOK) with a version of GROMACS [41] compiled in parallel such that each simulation is initiated from the final configuration of the closest T. 

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