ROLE OF THE LYSINE, TYROSINE AND TRYPTOPHAN RESIDUES IN THE ACTIVITY OF MILK LYSOZYMES

SINCE IT APPEARS that each amino ,acid in a protein chain has a specific role in the function of the molecule, it should be possible to elucidate the nature of this function by physical and chemical methods. The x-ray crystallographic studies of Blake et al. (1965, 1967) have yielded a three dimensional projection of egg white lysozyme (EC 3.2.1.17) and virtually every side chain residue which is susceptible to chemical modification has been studied in regards to its role in the conformation and activity of the enzyme. The lysine residues of egg white lysozyme (EWL) have been studied extensively but their role in the action of the enzyme is not yet clearly established. Chemical modifications such as guanidination which retain the positive charge of these residues do not affect lytic activity (Geschwind and Li, 1957; Parsons et al., 1969); whereas, modifications such as acetylation which remove the positive charge result in progressive inactivation at neutral pH (Parsons et al., 1969; Davies and Neuberger, 1969; Yamasaki et al., 1968a). Furthermore, the loss of lytic activity due to acetylation has been shown to be directly proportional to the number of acetyl groups introduced and the lowering of the isoelectric point of EWL. Complete acetylation of the lysozyme molecule does not seem to affect chitinase activity (Davies and Neuberger, 1969; Hayashi et al., 1968; Yamasaki et al., 196813). Atassi and Habeeb (1969) nitrated lysine residues 20 and 23 with tetranitromethane (TNM), and observed that the nitrated EWL possessed only 50% activity and subsequent reduction of the nitro group restored only slight activity. Both modifications caused conformational changes which were probably responsible for the loss in activity. The involvement of the tryptophan residues in the activity of EWL has been previously reported. Hayashi et al. (1963, 1964, 1965) noted that 80% of the lysozyme activity was lost upon oxidation of tryptophan 62 by N-bromosuccinimide (NBS). Hartdegen and Rupley (1964, 1967) found that iodine oxidation of tryptophan 108 destroyed most of the lytic activity of EWL. The crystallographic studies of Blake et al. (1965, 1967) and Phillips (1967) have confirmed the involvement of tryptophan residues 62 and 63 as well as tryptophan 108 in the substrate binding of EWL. Inhibition studies involving N-acetylglucosamine (NAG) and imidazole compounds have also shown the involvement of tryptophan in substrate binding of EWL. Neuberger and Wilson (1967) related changes in the tryptophan difference spectrum of lysozyme to the inhibitory power of NAG and its derivatives. Shinitzky et al. (1966a) observed that the fluorescence of lysozyme was affected by its binding with NAG and its polymers. They proposed that the spectral shifts were due to conformational changes of the protein that bring hydrophobic groups closer to the affected indole rings. Shinitzky et al. (1966b) also noted the formation of charge-transfer