Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada.
Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada.
J Inorg Biochem. 2018 May;182:1-8. doi: 10.1016/j.jinorgbio.2018.01.015. Epub 2018 Feb 2.
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 pH 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.
炭疽致死因子（LF）是一种锌依赖性内肽酶，在炭疽感染过程中，它参与切割对宿主信号通路维持至关重要的蛋白质。尽管锌通常被认为是体内的天然金属离子，但 LF 对金属取代具有很高的耐受性，其被铜取代后，酶的活性比天然锌蛋白（在 pH 7 时）高 4.5 倍。本研究表明，通过仔细选择缓冲液、离子强度、pH 值和底物，可以将 CuLF 和天然 LF 的活性比提高到>40 倍。使用荧光 LF 底物，可以利用这种优化的测定条件检测到低至 2 pM 的 LF 浓度。与锌形式不同，CuLF 被发现被溴化物和碘化物离子抑制，在酸性条件下不易失去金属，并且显示出明显的 pH 依赖性，碱性臂向更酸性条件移动。该酶的 pH 谱中的碱性臂被认为源自金属结合水分子的质子化状态的变化，该水分子在催化机制中充当亲核试剂。基于这些观察结果和对其他锌蛋白酶的研究，提出了 LF 的最小机制。
