Studies on metal exchange and inhibition in anthrax lethal factor

Abstract

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase, and a component of the anthrax toxin secreted by Bacillus anthracis. LF demonstrates two interesting contradictory features: a propensity to readily exchange the active site metal ion with extraneous metal ions in solution, while having at the same time a very high affinity (femto- to picomolar) for metals in its active site. The hypothesis of this thesis was that metal exchange in LF occurs via an associative mechanism involving the formation of a di-metal species through the occupation of an inhibitory metal-binding site (by the incoming metal) located adjacent to the active site. We demonstrate here that the spontaneous demetallation of LF is slow, especially for the release of zinc (t1/2 ~3 h). In contrast, metal exchange (with 70Zn2+, Co2+ and Cu2+) was found to occur with t1/2 values between 5 s and 180 s, hence supporting an associative mechanism of exchange. In addition, the mode of inhibition exerted by Zn2+ on zinc-containing LF (ZnLF) was observed to be non-competitive with an inhibition (pseudo)constant of ~30 μM, a feature in agreement with turnover studies using stopped-flow UV-Vis spectroscopy. Finally, present studies using Tb3+ suggest that the lanthanide ion competes with other metal ions (Zn2+, Cu2+) for binding to the inhibitory metal site in LF, hence decreasing the rate of metal exchange. This observation provides further support for the intimate link between a putative inhibitory binding site and metal exchange. The results of this study provide insight into how cellular metal pools may remain dynamic in vivo, and have implications not only for LF, but for many other zinc-dependent enzymes where metal substitution is used for spectroscopic studies.