Proteolysis of lysozyme-substrate complex

Lysozyme [EC 3. 2. 1. 17 mucopeptide muramylhydrolase] can form stable complexes with glycol chitin (substrate) and its partially and completely hydrolyzed products under appropriate conditions. On forming the complex with the substrate, the ultraviolet absorption band of lysozyme shifts toward longer wavelengths (Hayashi et al., 1963). This red-shift of the spectrum arises from the burial of a specific tryptophan residue of lysozyme into the interior of the molecule. The burial of the tryptophan residue is accompanied by a conformational change of whole molecule, and not due to mere covering of the tryptophan residue with the substrate* (Hayashi et al., 1964). The conformational changes accompanied by the formation of the enzyme-substrate cnmplexes have been observed with several enzymes by various physico-chemical techniques. Since the activity of a protease depends profoundly upon the conformation of the substrate proteins, the conformational changes in the enzyme molecule taking place on the formation of the enzyme-substrate complex can be followed with a change in the proteolytic digestibility of the enzyme molecule in the complex. Nirenberg and Jakoby (1960) reported that succinate semialdehyde dehydrogenase [EC 1.2.1.161 had increased sensitivity for tryptic digestion in the presence of the substrate. This finding was regarded as an induction of the conformational change of the enzyme by its substrate. It has long been known that the spectrum of a protein depends upon the environment of its chromophores ; for instance, the spectrum of tryptophan residues buried in an interior of the molecule is shifted toward longer wavelengths as compared to the residues accessible to medium. When such interior tryptophan residues are exposed to the medium by denaturation or by cleavage of peptide bonds, the spectrum shifts toward shorter wavelengths (blue-shift). The difference spectrum of a denatured protein which originally contained buried tryptophan residues exhibits a negative peak around 293 m/l. The blue-shift of lysozyme which closely resembled the blue-shift of tryptophan was first observed by Donovan et al. (1958). Lysozyme contains four tryptophan residues accessible to the medium and two inaccessible residues which are located in an interior of the molecule (Hamaguchi and Kurono, 1963). Inada (1961) reported that one of three tyrosine residues of lysozyme behaved abnormally and was normalized in the presence of 4 M guanidine hydrochloride. The contribution of the abnormal tyrosine residue to the blue-shift of lysozyme can be excluded by measuring the intensity of the main negative peak at 293 rnp, because the blue-shift differrence spectrum arisen from the tyrosine residue shows no intensity at 293 tnp, Laskowski et al. (1956) applied first the difference spectrophotometry to the tryptic digestion of insulin. Sela and Anfinsen (1957) studied the peptic digestion of ribonuclease with the same method and Bigelow and Ottesen (1957) applied this method to observe the abnormal tyrosine residues in pepsin-inactivated ribonuclease. The proteolysis of lysozyme seems to be followed accurately by difference spectrophotometry, owing to its high content of tryptophan residues in the interior of the molecule. The present paper deals with the digestibility of the lysozymesubstrate complex with proteases in connection with the conformational change