thromboplastin and 4-aminobenzamidine

Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca(2+) ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca(2+) and Mg(2+), and the Ca(2+) sites in FVIIa that could be specifically occupied by Mg(2+) are unknown. Furthermore, FVIIa contains a Na(+) and two Zn(2+) sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca(2+), Mg(2+), Na(+), and Zn(2+). The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca(2+) and three bound Mg(2+). The EGF1 domain contains one Ca(2+) site, and the protease domain contains one Ca(2+), one Na(+), and two Zn(2+) sites. (45)Ca(2+) binding in the presence/absence of Mg(2+) to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly(193) in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

Topics: Benzamidines; Catalysis; Factor VIIa; Humans; Models, Molecular; Peptide Mapping; Protein Conformation; Structure-Activity Relationship; Substrate Specificity; Thromboplastin

The prothrombinase complex, composed of factor Xa and factor Va assembled on a membrane surface, catalyzes the proteolytic formation of thrombin during blood coagulation. The molecular basis for the macromolecular substrate specificity of prothrombinase is poorly understood. By kinetic studies of prethrombin 2 cleavage by prothrombinase in the presence or absence of fragment 1.2, we show that occupation of the active site of the catalyst by inhibitors or alternate peptidyl substrates does not alter the affinity for prethrombin 2. Productive recognition of the macromolecular substrate therefore results from an initial interaction at enzymic sites (exosites) distinct from the active site, which largely determines substrate affinity. This interaction at exosites is evident even in the absence of activation peptide domains responsible for mediating the binding of the substrate to membranes or factor Va. Interactions at the active site with structures surrounding the scissile bond then precede bond cleavage and product release. The second binding step, which appears unfavorable, does not affect substrate affinity but contributes to the maximum catalytic rate. Therefore, binding specificity of prothrombinase for the macromolecular substrate is determined by exosites on the enzyme. We show that competitive inhibition of prethrombin 2 cleavage can be accomplished by interfering with the exosite binding step without obscuring the active site of the enzyme. These findings suggest limitations to the common approach of inferring the basis of factor Xa specificity with active site mutants or the targeting the active site of factor Xa with reversible inhibitors for therapeutic purposes. The achievement of distinctive macromolecular substrate specificities through exosite interactions and modulation of maximum catalytic rate through binding steps may also underlie the reactions catalyzed by the other coagulation complexes containing trypsin-like enzymes.

Topics: Animals; Benzamidines; Binding, Competitive; Cattle; Enzyme Precursors; Factor Va; Factor Xa; Factor Xa Inhibitors; Kinetics; Macromolecular Substances; Oligopeptides; Phenylmethylsulfonyl Fluoride; Prothrombin; Substrate Specificity; Thrombin; Thromboplastin