title: Lethal factor active-site mutations affect catalytic activity in vitro.
abstract: The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093-1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.
authors.affiliation: Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: S E
authors.initials: SE
authors.lastname: Hammond
authors.forename: P C
authors.initials: PC
authors.lastname: Hanna
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011480:Protease Inhibitors; C030325:anthrax toxin; D010450:Endopeptidases