Substrate recognition of anthrax lethal factor examined by combinatorial and pre-steady-state kinetic approaches.
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.
Zakharova
Maria Yu
MY
M. M. Shemyakin & Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997.
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
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics* / Q000378:metabolism*; D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain / Q000502:physiology; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli; D006868:Hydrolysis; D007700:Kinetics; D048669:MAP Kinase Kinase 6 / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D013379:Substrate Specificity
D000942:Antigens, Bacterial; D001051:Apoenzymes; D001427:Bacterial Toxins; D002413:Cations, Divalent; D019151:Peptide Library; C030325:anthrax toxin; D048669:MAP Kinase Kinase 6