SITE-DIRECTED MUTAGENESIS OF HISTIDINE-RESIDUES IN CLOSTRIDIUM-PERFRINGENS ALPHA-TOXIN

Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin res ulted in complete loss of hemolytic, phospholipase C, sphingomyelinase , and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the acti vities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 s howed no effect on the biological activities, indicating that these re sidues are not essential for these activities. The variant toxin at H- 11 was not detect;ed in culture supernatant and in cells of the transf ormant carrying the variant toxin gene. Wild-type toxin and the varian t toxin at H-148 bound to erythrocytes in the presence of Ca2+; howeve r, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ i ons stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence, of Ca2+ and caused an increase in hemolyti c activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactiv ated by EDTA contained two zinc atoms. These results suggest that wild -type toxin contains two tightly bound zinc atoms which are not coordi nated to H-68, -126, and -136. The variant toxin at H-148 possessed on ly one zinc atom. Wild-type toxin and the variant toxin at H-148 showe d [Zn-65](2+) binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [Zn-65](2+) binding to wild-type toxin was compe titively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results su ggest that H-68, -126, and -136 residues bind an exchangeable and labi le metal which is important for binding to membranes and that H-148 ti ghtly binds one Zinc atom which is essential for the active site of al pha-toxin.