Replacement of Phe274 With Conserved Residue Tyr274 for Reactive Center Loop Expulsion in Antithrombin

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 (K_D; 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.

Keywords

antithrombin reactive center loop serpin

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