Replacement of Phe274 With Conserved Residue Tyr274 for Reactive Center Loop Expulsion in Antithrombin
- Duhan, Urmila
Abstract
Background:
The reactive center loop (RCL) of native antithrombin is partially inserted in the main serpin body. It must be fully exposed for optimal inhibitory function.
Objective:
To test the hypothesis that P14-s2B interaction affects loop insertion in antithrombin. By mutating Phe274 to Tyr274, the objective was to introduce P14-s2B interaction in antithrombin.
Methods:
Site-directed mutagenesis and affinity chromatography were used to obtain purified recombinant protein. Antithrombin's ability to form sodium dodecyl sulfate (SDS)-stable complex with thrombin, stoichiometry of thrombin inhibition, second-order rate constant for thrombin and factor Xa (fXa) inhibition (M-1 s-1), and heparin dissociation constant (KD; tryptophan fluorescence emission spectra) were determined.
Results and Conclusion:
A marginal, but inconclusive, difference between the wild type and the mutant was observed. The result highlights the variable effect of P14-s2B interaction in different serpins. Alternate hypothesis for achieving loop expulsion is proposed.