Binding of divalent copper ions to aspartic acid residue 52 in hen egg-white lysozyme.

Divalent copper was found to inhibit non-competitively the lysis of Micrococcus lysodeikticus cells by hen egg-white lysozyme, with an inhibition constant K, = 3.8 X lo2 M-l. The association constants of Cu2+ for lysozyme and for a derivative of lysozyme in which tryptophan residue 108 was selectively modified, were measured spectrofluorimetrically and found to be 1.8 X lo2 M-l and 1.0 X lo3 M-l, 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 A from the carboxyl side-chain of aspartic acid 52,5 A from the carboxyl of glutamic acid 35 and about 7 A from tryptophan 108. The addition of a saccharide inhibitor to lysozyme was found to increase the association constant of Cu 2+ for lysozyme from a value of 1.8 x lo2 M-l to 6.0 x lo2 M-l. This finding was interpreted as indicative of a change in conformation around tryptophrtn 108 and glutamic acid 35 induced by the interaction of ssccharides with the enzyme, which affects the metal binding properties of aspartic acid 52.