title: Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate.
abstract: The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex.
authors.affiliation: Joseph Stokes Research Institute, Children's Hospital of Philadelphia, and the Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Orcutt
authors.forename: Concetta
authors.initials: C
authors.lastname: Pietropaolo
authors.forename: Sriram
authors.initials: S
authors.lastname: Krishnaswamy
mesh_terms: D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D011516:Prothrombin / Q000378:metabolism; D013379:Substrate Specificity; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D011516:Prothrombin; D013925:Thromboplastin