Ozone with lysozyme, reaction

Leh, F.. and 1. B. Mudd. Reaction of ozone with lysozyme, pp. 22-39. In M. Dugger, Ed. Air Pollution Effects on Plant Growth. ACS Symposium Series 3. Washington. D.C. American Chemical Society, 1974. 

D14. Dooley, M. M., and Mudd, J. B., Reaction of ozone with lysozyme under different exposure conditions. Arch. Biochem. Biophys. 218, 459-471 (1982). 

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. 

Distinct changes in several properties of lysozyme occur after reaction with ozone. The lytic activity of the ozonized lysozyme shows the same trend at various pHs as the native enz3mie (Fig. 2) this may suggest that the pK values of the ionizable groups involved in catalysis have not been altered by ozonplysis. The amino acid composition of ozonized lysozyme differs from that of the native enz3mie in three residues — methionine, tryptophan and t3H osine. None of the other amino acids is affected by ozone. The extensive loss of enz5miic activity must be ascribed to the oxidative modification of these three amino acid residues in the lysozyme. 

In an earlier experiment, Jori et al. (14) reported that methionyl residues are important in maintaining the tertiary structure of lysozyme. The introduction of a polar center into the aliphatic side chain of methionine, as a consequence of the conversion of the thioether function to the sulfoxide, may bring about a structural change of the lysozyme molecule which, in turn, reduces the catalytic efficiency. When ozonized lysozyme was treated with 2-mercaptoethanol in an aqueous solution according to the procedure of Jori e al. (14), the enzyme did not show any increase in its activity. This may be explained in two ways. In one, such reactions are complicated by many side reactions, e.g. sulfhydryl-disulfide interchange, aggregation and precipitation of the modified enzyme (24-26). In the other, the failure to recover the activity of the enzyme may by associated with the extensive oxidation of other residues. 

Chicken egg white lysozyme (LZM) does not possess exposed methionyl residues, and it has six tryptophan residues, three of which, located at positions 62,108, and 111 are readily oxidizable with ozone and are built-in the LZM active center (D14). Tryptophanyl residues are also the first reacting moieties upon treatment of LZM with the MPO-Cr-H2C>2 system (at pH 4.5). The reaction occurs in several stages. In the first stage, which occurs when 1.4-1.8 mol of H2O2 for 1 mol of LZM is used, LZM loses its enzyme activity, but no derivative distinguishable from the native protein on the polyacrylamide gel electrophoresis is formed. The inactivation may be prevented by addition to the reaction medium A-acetylcysteine or... 

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