The co‐crystal structure of unliganded bovine α‐thrombin and prethrombin‐2: Movement of the Tyr‐Pro‐Pro‐Trp segment and active site residues upon ligand binding

Unliganded bovine α‐thrombin and prethrombin‐2 have been co‐crystallized, in space group P21212, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 Å and 2.3 Å, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (Rfree = 0.255) and 0.190 (Rfree = 0.282) for the salt and PEG2K models, respectively. The apo‐enzyme form of bovine α‐thrombin shows dramatic shifts in placement for the Tyr‐Pro‐Pro‐Trp segment, for Glu‐192, and for the catalytic residues His‐57 and Ser‐195, when compared to 4 thrombin complexes representing different states of catalysis, namely (1) the Michaelis complex (residues 7‐19 of fibrinogen Aa with a non‐cleavable scissile bond), (2) enzyme‐inhibitor complex (D‐Phe‐Pro‐Arg chloromethylketone), (3) enzyme product complex (residues 7‐16 of fibrinopeptide A), and (4) the exosite complex (residues 53‐64 of hirudin). The structures of bovine and human prethrombin‐2 are generally similar to one another (RMS deviation of 0.68 8,) but differ significantly in the Arg‐15/Ile‐16 cleavage region and in the three activation domains, which are disordered in bovine prethrombin‐2, analogous to that seen for trypsinogen.