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Substrate recognition of anthrax lethal factor examined by combinatorial and pre-steady-state kinetic approaches.

ABSTRACT: Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca(2+) and Mn(2+). Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.

SUBMITTER: Zakharova MY

PROVIDER: S-EPMC2709388 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Substrate recognition of anthrax lethal factor examined by combinatorial and pre-steady-state kinetic approaches.

Zakharova Maria Yu MY Kuznetsov Nikita A NA Dubiley Svetlana A SA Kozyr Arina V AV Fedorova Olga S OS Chudakov Dmitry M DM Knorre Dmitry G DG Shemyakin Igor G IG Gabibov Alexander G AG Kolesnikov Alexander V AV

The Journal of biological chemistry 20090409 27

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Pha ...[more]

PMID: 19359249

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Substrate recognition of anthrax lethal factor examined by combinatorial and pre-steady-state kinetic approaches.

Publications

Substrate recognition of anthrax lethal factor examined by combinatorial and pre-steady-state kinetic approaches.

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