Lysozyme site selection

Estimation of the Binding Site. Tryptophan-108 shows a specific reaction with iodine, distinguishing it from other tryptophan residues of lysoz3mie. When try-108 is selectively oxidized by iodine, lysozyme completely loses its activity. Nevertheless, the lysozyme still shows the ability to form an enzyme-substrate complex with CM-chitin. This observation contributes to the conclusion that try-62 is an essential binding site for a complex formation (13). All ozonized lysozymes formed strong complexes with CM-chitin and could only be eluted by 0.2N HA (Fig. 6). This further confirms that two tryptophan residues (108 and 111) are indispensible for the hydrolytic action of lysozyme, and that inactivation by ozone cannot be attributed to inhibition of substrate binding capability. 

Aliphatic hydroxyl groups cannot normally be selectively modified except in certain special cases such as the serine proteinases. In anhydrous formic acid, the A,O-acyl migration that occurs in strong sulfuric or phosphoric acid apparently does not occur. Instead there is formylation of the serine and threonine residues (208). Enzymically inactive aggregates are produced, but the reaction is reversed in aqueous solution at neutral pH and the activity returns. Josefsson reported the introduction of 29 formyl groups in RNase (209) as compared to the total of 25 Ser and Thr residues. This identification of reaction sites is not clear, however, since the number of formyl groups introduced into lysozyme far exceeded the Ser-Thr total. 

Figure 9.3. Restriction map produced by Webcutter. The partial restriction map shows the nucleotide sequence of human lysozyme gene submitted to Webcutter using options for all restriction endonucleases with recognition sites equal to or greater than six nucleotides long and cutting the sequence 2 6 times (at least 2 times and at most 6 times). The restriction profile (map) is returned if Map of restriction sites is selected for display. The tables by enzyme name and by base pair number can be also returned if displays for Table of sites, sorted alphabetically by enzyme name Table of sites, sorted sequentially by base pair number are chosen.

Figure 15.5. Docking by merging files with SPDBV. The docking of trisaccharide (NAM-NAG-NAM) into the active site of lysozyme by merging trisaccharide file (9lyz.pdb) onto lysozyme file (llyz.pdb). After the merge, only active-site residues (catalytic residues in green and contact residues in blue) and trisaccharide (in red) are selected to display and saved.

Fig. 10.T. Selective ion binding by human lysozyme upper left, wild type upper middle, Q86D upper right, A92D lower left, Q86D/A92D. The lower middle picture shows the calcium binding site in the Q86D/A92D mutant detected by X-ray, while the picture in lower right exhibits the binding-site found by the 3D-RISM theory...

Axial symmetry cannot be assumed as in the case of lysozyme, since data are available only for one lanthanide ion. Data with other lanthanide ions were not obtained because selectivity of the EF site over the CD site varies with increase in cation size and hence it is not possible to test for axial symmetry. 

A carboxyethyl derivative of lysozyme and synthetic disulphide peptides have been used to study the enzymic and immunochemical properties of lysozyme around the disulphide bridge (linking residues 6—127) at the reactive site. One of the six tryptophanyl residues, possibly either tryptophan-62 or -63, of hen egg-white lysozyme has been oxidized selectively with ozone in aqueous solution. N -Formylkynurenine was formed, with concomitant loss of the enzymic activity, although there was apparently little change in the gross conformation of the molecule. 

High-resolution n.m.r. combined with information from X-ray diffraction, is revealing much about the active sites and mechanisms of action of various enzymes, such as chymotrypsin (Gerig, 1968) and lysozyme (Cohen and Jardetzky, 1968), and the same benefits are expected for drugs and their receptors. Selective deuteration of the enzyme is a great help in these studies (Putter et al.y 1969). 

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