Divalent copper was found to inhibit non-competitively the lysis of Micrococcus lysodeikticus cells by hen egg-white lysozyme, with an inhibition constant Ka= 3.8 × 102m-1. The association constants of Cu2+for lysozyme and for a derivative of lysozyme in which tryptophan residue 108 was selectively modified, were measured spectrofluorimetrieally and found to be 1.8 × 102m-1and 1.0 × 103m-1, respectively. The electron spin resonance spectrum of Cu2+was not affected by the addition of lysozyme, whereas many new lines appeared on addition of the modified protein. This was interpreted as evidence for the binding of Cu2+in the neighbourhood of tryptophan 108. To unequivocally establish the site of ligation of Cu2+, crystals of lysozyme soaked in Cu2+were examined by X-ray crystallography and the results compared to those obtained from crystals of native lysozyme. Cu2+was found to be located 2 to 3 Å from the carboxyl side-chain of aspartic acid 52, 5 Å from the carboxyl of glutamic acid 35 and about 7 Å from tryptophan 108. The addition of a saccharide inhibitor to lysozyme was found to increase the association constant of Cu2+for lysozyme from a value of 1.8 × 102m-1to 6.0 × 102m-1. This finding was interpreted as indicative of a change in conformation around tryptophan 108 and glutamic acid 35 induced by the interaction of saccharides with the enzyme, which affects the metal binding properties of aspartic acid 52. © 1974.
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