Stability effects associated with the introduction of a partial and a complete Ca2+-binding site into human lysozyme

Two mutants of human lysozyme were synthesized. Mutant A92D,in which Ala92 was substituted by Asp, contains a partial Ca²⁺-bindingsite and mutant M4, in which Ala83, Gm86, Asn88 and Ala92 werereplaced by Lys, Asp, Asp and Asp respectively, contains thecomplete Ca -binding site of bovine a-lactalbumin. The Ca²⁺-bindingconstants of wild type human lysozyme and of mutants A92D andM4, measured at 25C and pH 7.5, were 2(1) x 102 M"1, 8(2)x l^M"1 and 9(0.5) x 10* M"1 respectively. Information gatheredfrom mkrocalorimetrk and CD spectro-scopic measurements indicatesthat the conformational changes of the M4 mutant lysozyme, inducedby Ca²⁺ binding, are smaller than those observed for bovinea-lactalbumin and for the Ca²⁺-binding equine lysozyme. At pH4.5, the thermostability of both the apo and Ca²⁺ forms of theA92D human was decreased in comparison with that of native humanlysozyme. In particular, within the apo form of this mutantan a-helix-containing sequence was destabilized. In contrast,at the same pH the thermostability of the apo and Ca²⁺ formsof the M4 mutant lysozyme was increased. The e-ammonium groupof the Lys83 side chain is assumed to be responsible for thestabilization of the apo form of this mutant.