title: Site-directed mutagenesis of the leech-derived factor Xa inhibitor antistasin. Probing of the reactive site.
abstract: Antistasin (ATS) is a leech-derived 119-amino-acid protein which exhibits potent and highly selective inhibition of coagulation Factor Xa. It inhibits Factor Xa according to a common mechanism of serine-proteinase inhibitors in which a conformationally rigid substrate-like reactive site is presented to the enzyme. In this study a recombinant version of ATS was expressed and purified utilizing a yeast expression system in order to probe the reactive site P1 (Arg-34) and P1' (Val-35) residues by site-directed mutagenesis. The results demonstrate the requirement for a positively charged residue in the P1 position of ATS, with an arginine residue preferred over a lysine, yielding K1 values of 61 pM and 1.28 nM respectively. Mutation of the P1 arginine residue to the non-polar amino acid leucine abolished its inhibitory potency toward Factor Xa. The role of the C-terminal domain of ATS, which shares significant amino acid sequence identity with the N-terminal domain, was investigated by creating a second reactive site in the corresponding position of the C-terminal domain. The inhibitory activity of this mutant demonstrated that the C-terminal domain of ATS is not folded into the proper conformation necessary to create a functional inhibitory domain.
authors.affiliation: Department of Cellular and Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.
authors.forename: K J
authors.initials: KJ
authors.lastname: Hofmann
authors.forename: E M
authors.initials: EM
authors.lastname: Nutt
authors.forename: C T
authors.initials: CT
authors.lastname: Dunwiddie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001780:Blood Coagulation Tests; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D065427:Factor Xa Inhibitors; D006801:Humans; D007447:Invertebrate Hormones / Q000235:genetics*; D007700:Kinetics; D007865:Leeches; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid
chemical_list: D065427:Factor Xa Inhibitors; D007447:Invertebrate Hormones; D011994:Recombinant Proteins; C053155:antistasin; D004247:DNA