26th Annual Student Research Conference
Thrombin mutant G216A provides insight into mechanism of E-E* structure transition
Lisa J. Clark
Dr. Cynthia Cooper, Faculty Mentor
Thrombin plays a vital role in blood coagulation. The serine protease cleaves procoagulant substrates fibrinogen and protease activated receptor 1 (PAR1) and also activates anticoagulant protein C upon binding of thrombomodulin. Thrombin has three forms: E* (anticoagulant, W215 side chain flip and significant active site occlusion by residues 215-217), E (lowered activity), E:Na+ (fully active). X-ray crystallographic structure of thrombin mutant G216A demonstrates an intermediate structure in E-E* transition: partial active site occlusion (21.4%) and W215 side chain flip. G216A exhibits greater reduction of kcat/Km values for PAR1 and fibrinogen than that for protein C, designating the mutant as anticoagulant. G216A, crystallized in absence of allosteric Na+, displays E192-G193 peptide bond flip that disrupts the oxyanion hole (amido hydrogens of G193 and S195 that stabilize tetrahedral intermediate of peptide hydrolysis) required for optimum kinetic activity. Comparison of other previously crystallized E forms and G216A indicates intrinsic flexibility of E192-G193 bond.
Keywords: Thrombin, x-ray crystallography, enzyme kinetics
Topic(s):Biology
Presentation Type: Poster
Session: 2-1
Location: GEO
Time: 3:30