Lysozyme active site

Hen egg-white lysozyme catalyzes the hydrolysis of various oligosaccharides, especially those of bacterial cell walls. The elucidation of the X-ray structure of this enzyme by David Phillips and co-workers (Ref. 1) provided the first glimpse of the structure of an enzyme-active site. The determination of the structure of this enzyme with trisaccharide competitive inhibitors and biochemical studies led to a detailed model for lysozyme and its hexa N-acetyl glucoseamine (hexa-NAG) substrate (Fig. 6.1). These studies identified the C-O bond between the D and E residues of the substrate as the bond which is being specifically cleaved by the enzyme and located the residues Glu 37 and Asp 52 as the major catalytic residues. The initial structural studies led to various proposals of how catalysis might take place. Here we consider these proposals and show how to examine their validity by computer modeling approaches. 

FIGURE 6.6. The type of model compounds that were used to estimate the electrostatic stabilization in lysozyme (the only hydrogen atom shown, is the one bonded to the oxygen). Such molecules do not show a large rate acceleration due to electrostatic stabilization of the positively charged carbonium transition state. However, the reaction occurs in solution and not in a protein-active site, and the dielectric effect is expected to be very different in the two cases. 

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. 

FIGURE 6.11. Comparison of the environment around the transition state of lysozyme in the enzyme-active site and in the reference solvent cage. 

Enzyme active sites, 136,148, 225. See also Protein active sites in carbonic anhydrase, 197-199 in chymotrypsin, 173 in lysozyme, 153, 157 nonpolar (hypothetical site), 211-214 SNase, 189-190,190 steric forces in, 155-158, 209-211, 225 in subtilisin, 173 viewed as super solvents, 227 Enzyme cofactors calcium ... 

LD model, see Langevin dipoles model (LD) Linear free-energy relationships, see Free energy relationships, linear Linear response approximation, 92,215 London, see Heitler-London model Lysine, structure of, 110 Lysozyme, (hen egg white), 153-169,154. See also Oligosaccharide hydrolysis active site of, 157-159, 167-169, 181 calibration of EVB surfaces, 162,162-166, 166... 

From crystal-structure analysis of hen-egg lysozyme and of its complex with the competitive inhibitor tri-Af-acetylchitotriose, the following conclusions were drawn the active site consists of a cleft containing six sub-sites, A to F, of which each could accommodate a) -( 1 — 4)-linked A-acetylglucosa-... 

Finally, we come to enzyme models. D. W. Griffiths and M. L. Bender describe the remarkable catalytic property of certain cycloamyloses which act through formation of inclusion complexes, and in this respect recall the clefts containing the active sites in enzymes such as lysozyme and papain. 

Following myoglobin and lysozyme, bovine carboxypeptidase A was the third protein to have its 3-D structure solved at high resolution. The active site zinc is bound to His69, Glu72 and Hisl96 (Figure 12.4), and to a water molecule, which is displaced when a... 

Figure 12.8 Some other active-site coordination motifs in mononuclear zinc enzymes from left to right bacteriophage T7 lysozyme, 5-aminolaevulinate dehydratase, Ada DNA repair protein. (Reprinted with permission from Parkin, 2004. Copyright (2004) American Chemical Society.)...

Figure 3. Stereo view of the active site cleft of lysozyme near site D. Hydrogen bonds of the Glu 35 sidechain are shown in dotted lines. The sidechain atom Hei of Glu 35 is shown.

Tryptophan 108 is recognized to be an active site in promoting the hydrolysis of 3(l,4)-glycosidic linkages between amino sugar residues in polysaccharide components of the bacterial cell walls. This residue is shown to occupy the cleft as well as trjrptophan 62 and 63, and is in a hydrophobic region. Tryptophan residues 62 and 108 are indispensable for the action of lysozyme, and tryptophan 62 is known to be the only binding site for the complex formation (13). Oxidation of tryptophan-108 is expected... 

D. The binding of polysaccharide substrates that have six or more sugar groups to lysozyme, the enzyme in tears and saliva that cleaves such molecules, induces strain in the sugar nearest the active site making the nearby bond more susceptible to hydrolysis. 

A good example of this interaction in catalysis is the hydrolysis of the bacterial cell wall polysaccharide by lysozyme. This enzyme contains two carboxylic gronps at its active site and, in active enzyme one must be in dissociated—COO, the other in the undissociated—COOH form. Therefore, the pK s of the two carboxylic groups ate different. This difference in dissociation constant is a consequence of the neighbouring amino acid residues and of the interactions between the functional groups in the microenvironment. 

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