RESUMO
Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of proteins critical to the maintenance of host signaling pathways during anthrax infections. Although zinc is typically regarded as the native metal ion in vivo, LF is highly tolerant to metal substitution, with its replacement by copper yielding an enzyme (CuLF) 4.5-fold more active than the native zinc protein (at pHâ¯7). The current study demonstrates that by careful choice of the buffer, ionic strength, pH and substrate, the activity ratio of CuLF and native LF can be increased to >40-fold. Using a fluorogenic LF substrate, such optimized assay conditions can be exploited to detect LF concentrations as low as 2 pM. In contrast to the zinc form, CuLF was found to be inhibited by bromide and iodide ions, to be resistant to metal loss under acidic conditions, and to display a sharp pH dependence with significantly shifted alkaline limb towards more acidic conditions. The alkaline limb in the enzyme's pH profile is suggested to originate from changes in the protonation state of the metal-bound water molecule which serves as the nucleophile in the catalytic mechanism. Based on these observations and studies on other zinc proteases, a minimal mechanism for LF is proposed.