NDLI: Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches*

Content Provider	PubMed Central
Author	Zakharova, Maria Yu Kuznetsov, Nikita A. Dubiley, Svetlana A. Kozyr, Arina V. Fedorova, Olga S. Chudakov, Dmitry M. Knorre, Dmitry G. Shemyakin, Igor G. Gabibov, Alexander G. Kolesnikov, Alexander V.
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 Ca2+ and Mn2+. 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.
Related Links	http://dx.doi.org/10.1074/jbc.M807510200
Starting Page	17902
File Format	PDF
ISSN	1083351X
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	27
Volume Number	284
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2009-07-03
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

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