Alkaline earth metals are not required for the restoration of the apoform of anthrax lethal factor to its holoenzyme state

Anthrax lethal factor (LF) is a zinc-dependent metalloendopeptidase previously shown to require calcium and magnesium for the restoration of its catalytic function upon exposure of the apoprotein (apoLF) to Zn²⁺. Since concrete Ca²⁺/Mg²⁺ binding sites have not been identified in LF, the effects of alkaline earth metals on the enzymatic function of holoLF (ZnLF) and on the reconstitution of apoLF were reinvestigated. The current study reveals alkaline earth metals to be inhibitory at concentrations higher than 1 mM. A combination of activity/inhibition assays and Tb³⁺ luminescence spectroscopy was employed to unequivocally establish the presence of at least one inhibitory low-affinity Ca²⁺-site in LF. A comparative analysis of apoLF preparations obtained by dialysis and centrifugal filtration (following treatment of ZnLF with chelators) revealed the exposure of apoLF to low equimolar amounts of Zn²⁺ to be sufficient for the full restoration of the protein’s catalytic competence, a finding constistent with the picomolar dissociation constant of ZnLF determined in this study. The previously documented requirement of Ca²⁺ and Mg²⁺ in apoLF reconstitution may be explicable on the basis of contamination of dialyzed apoprotein preparations with residual chelator, a phenomenon not encountered with apoLF obtained by centrifugal filtration.